Liquid adenovirus formulations

ABSTRACT

The present invention addresses the need to improve the long-term storage stability (i.e. infectivity) of vector formulations. In particular, it has been demonstrated that for adenovirus, the use of bulking agents, cryoprotectants and lyoprotectants imparts desired properties that allow both lyophilized and liquid adenovirus formulations to be stored at 4° C. for up to 6 months and retain an infectivity between 60-100% of the starting infectivity.

This application is a continuation of U.S. patent application Ser. No.09/941,296, filed Aug. 28, 2001 (now issued as U.S. Pat. No. 7,235,391),which is a divisional of U.S. patent application Ser. No. 09/441,410,filed Nov. 16, 1999 (now issued as U.S. Pat. No. 6,689,600), whichclaims the benefit of U.S. Provisional Patent Application Ser. No.60/133,116, filed May 7, 1999, and U.S. Provisional Patent ApplicationSer. No. 60/108,606, filed Nov. 16, 1998, the entire contents of theseapplications are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

A. Field of the Invention

The present invention relates generally to the fields of molecularbiology, virus production and gene therapy. More particularly, itconcerns methods for the formulation of highly purified lyophilized andliquid adenovirus particles stable for long-term storage. An importantembodiment of the present invention is the use of such long-term storagevirus preparations for gene therapy treatments of viral disease, geneticdisease and malignancies.

B. Description of Related Art

Viruses are highly efficient at nucleic acid delivery to specific celltypes, while often avoiding detection by the infected hosts immunesystem. These features make certain viruses attractive candidates asgene-delivery vehicles for use in gene therapies (Robbins andGhivizzani; 1998; Cristiano et al., 1998). Retrovirus, adenovirus,adeno-associated virus (AAV), and herpes simplex virus are examples ofcommonly used viruses in gene therapies. Each of the aforementionedviruses has its advantages and limitations, and must therefore beselected according to suitability of a given gene therapy (Robbins andGhivizzani; 1998).

A variety of cancer and genetic diseases currently are being addressedby gene therapy. Cardiovascular disease (Morishita et al., 1998),colorectal cancer (Fujiwara and Tanaka, 1998), lung cancer (Roth et al.,1998), brain tumors (Badie et al., 1998), and thyroid carcinoma (Braidenet al., 1998) are examples of gene therapy treatments currently underinvestigation. Further, the use of viral vectors in combination withother cancer treatments also is an avenue of current research (Jounaidiet al., 1998).

Viral particles must maintain their structural integrity to beinfectious and biologically active. The structural integrity of a viralvector often is compromised during the formulation process, thusprecluding its use as a gene therapy vector. Adenoviruses for genetherapy traditionally have been formulated in buffers containing 10%glycerol. Formulated adenovirus is stored at <−60° C. to ensure goodvirus stability during storage. This ultra-low temperature storage notonly is very expensive, but creates significant inconvenience forstorage, transportation and clinic use. There is an urgent need todevelop new formulation for adenovirus that can be stored atrefrigerated condition.

Lyophilization has been used widely to improve the stability of variousviral vaccine and recombinant protein products. It is expected that thelong-term storage stability of adenovirus can be improved by reducingthe residual water content (moisture) in the formulated product throughlyophilization. However, there have not been reported studies on thelyophilization of live adenovirus for gene therapy.

Generally it is assumed that adenovirus will not maintain itsinfectivity when stored at refrigerated condition in a liquid form forextended period of time. As a result, there are no reported studies onformulating and storing adenovirus at refrigerated condition in a liquidform. Thus, there remains a need for long-term storage stableformulations of viral preparations.

SUMMARY OF THE INVENTION

The present invention addresses the need for improved, storage stableviral formulations, and methods for the production thereof, for use ingene therapy. In particular embodiments, a pharmaceutical adenoviruscomposition comprising adenovirus particles and pharmaceuticalexcipients, the excipients including a bulking agent and one or moreprotectants, wherein the excipients are included in amounts effective toprovide an adenovirus composition that is storage stable. In preferredembodiments, the adenovirus composition has an infectivity of between 60and 100% of the starting infectivity, and a residual moisture of lessthan about 5%, when stored for six months at 4° centigrade.

In one embodiment, the adenovirus composition is a freeze driedcomposition. In particular embodiments, the bulking agent in the freezedried adenovirus composition forms crystals during freezing, wherein thebulking agent is mannitol, inositol, lactitol, xylitol, isomaltol,sorbitol, gelatin, agar, pectin, casein, dried skim milk, dried wholemilk, silicate, carboxypolymethylene, hydroxyethyl cellulose,hydroxypropyl cellulose, hydroxypropyl methylcellulose ormethylcellulose.

In certain embodiments, the bulking agent in the freeze dried adenoviruscomposition is mannitol. In other embodiments the composition is furtherdefined as an aqueous composition comprising mannitol in a concentrationof from about 1% to about 10% (w/v). In another embodiment, the aqueouscomposition comprises the mannitol in a concentration of from about 3%to 8%. In a preferred embodiment, the aqueous composition comprisesmannitol in a concentration of from about 5% to 7%.

In certain embodiments, the freeze dried composition is prepared from anaqueous composition comprising a bulking agent in a concentration offrom about 1% to 10% (w/v). In other embodiments the freeze driedcomposition is prepared from an aqueous composition comprising a bulkingagent in a concentration of from about 3% to 8%. In yet otherembodiments, the freeze dried composition is prepared from an aqueouscomposition comprising a bulking agent in a concentration of from about5% to 7%.

In particular embodiments, pharmaceutical excipients serve as aprotectants. In one embodiment, the protectant is further defined as acryoprotectant. In certain embodiments, the cryoprotectant is anon-reducing sugar. In particularly defined embodiments the non-reducingsugar is sucrose or trehalose. In preferred embodiments the non-reducingsugar is sucrose.

In certain embodiments, the composition is further defined as an aqueouscomposition comprising a non-reducing sugar in a concentration of fromabout 2% to about 10% (w/v). In other embodiments, the aqueouscomposition comprises the sugar in a concentration of from about 4% to8%. In still other embodiments, the aqueous composition comprises thesugar in a concentration of from about 5% to 6%.

In one embodiment, the freeze dried composition is prepared from anaqueous composition comprising a non-reducing sugar in a concentrationof from about 2% to 10% (w/v). In other embodiments, the freeze driedcomposition is prepared from an aqueous composition comprising anon-reducing sugar in a concentration of from about 4% to 8%. In yetother embodiments, the freeze dried composition is prepared from anaqueous composition comprising a non-reducing sugar in a concentrationof from about 5% to 6%.

In another embodiment, the cryoprotectant is niacinamide, creatinine,monosodium glutamate, dimethyl sulfoxide or sweet whey solids.

In certain embodiments, the protectant includes a lyoprotectant, whereinthe lyoprotectant is human serum albumin, bovine serum albumin, PEG,glycine, arginine, proline, lysine, alanine, polyvinyl pyrrolidine,polyvinyl alcohol, polydextran, maltodextrins,hydroxypropyl-beta-cyclodextrin, partially hydrolysed starches, Tween-20or Tween-80. In a preferred embodiment, the lyoprotectant is human serumalbumin.

In certain embodiments, the composition is further defined as an aqueouscomposition comprising the lyoprotectant in a concentration of fromabout 0.5% to about 5% (w/v). In another embodiment, the aqueouscomposition comprises the lyoprotectant in a concentration of from about1% to about 4%. In still another embodiment, the aqueous compositioncomprises the lyoprotectant in a concentration of from about 1% to about3%.

In particular embodiments, the freeze dried composition is prepared froman aqueous composition comprising a lyoprotectant in a concentration offrom about 0.5% to 5% (w/v). In other embodiments, the freeze driedcomposition is prepared from an aqueous composition comprising alyoprotectant in a concentration of from about 1% to 4%. In anotherembodiment, the freeze dried composition is prepared from an aqueouscomposition comprising a lyoprotectant in a concentration of from about1% to 3%.

In one embodiment, pharmaceutical excipients defined as protectants,comprise both a lyoprotectant and a cryoprotectant.

Also contemplated in the present invention is an aqueous pharmaceuticaladenovirus composition comprising a polyol in an amount effective topromote the maintenance of adenoviral infectivity. In one embodiment,adenoviral infectivity of the adenovirus polyol composition is furtherdefined as maintaining an infectivity of about 70% PFU/mL to about 99.9%PFU/mL of the starting infectivity when stored for six months at 4°centigrade. In preferred embodiments, adenoviral infectivity is about80% to 95% PFU/mL of the starting infectivity when stored for six monthsat 4° centigrade.

In the context of the present invention, a polyol is defined as apolyhydric alcohol containing two or more hydroxyl groups. In certainembodiments, the polyol is glycerol, propylene glycol, polyethyleneglycol, sorbitol or mannitol, wherein the polyol concentration is fromabout 5% to about 30% (w/v). In other embodiments, the polyolconcentration is from about 10% to about 30%. In yet other embodiments,the polyol concentration is about 25%.

In a preferred embodiment, the aqueous pharmaceutical adenoviruscomposition comprises a polyol in an amount effective to promote themaintenance of adenoviral infectivity, wherein the polyol is glycerol,included in a concentration of from about 5% to about 30% (w/v).

In other embodiments, the aqueous pharmaceutical adenovirus compositioncomprising a polyol in an amount effective to promote the maintenance ofadenoviral infectivity further comprises an excipient in addition to thepolyol, wherein the excipient is inositol, lactitol, xylitol, isomaltol,gelatin, agar, pectin, casein, dried skim milk, dried whole milk,silicate, carboxypolymethylene, hydroxyethyl cellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, methylcellulose, sucrose,dextrose, lactose, trehalose, glucose, maltose, niacinamide, creatinine,monosodium glutamate dimethyl sulfoxide, sweet whey solids, human serumalbumin, bovine serum albumin, PEG, glycine, arginine, proline, lysine,alanine, polyvinyl pyrrolidine, polyvinyl alcohol, polydextran,maltodextrins, hydroxypropyl-beta-cyclodextrin, partially hydrolysedstarches, Tween-20 or Tween-80.

In further defined embodiments, the aqueous pharmaceutical adenoviruscomposition comprising a polyol further comprises in addition to thepolyol at least a first and a second excipient, wherein the secondexcipient is different the first excipient, and the excipient isinositol, lactitol, xylitol, isomaltol, gelatin, agar, pectin, casein,dried skim milk, dried whole milk, silicate, carboxypolymethylene,hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, methylcellulose, sucrose, dextrose, lactose, trehalose,glucose, maltose, niacinamide, creatinine, monosodium glutamate dimethylsulfoxide, sweet whey solids, human serum albumin, bovine serum albumin,PEG, glycine, arginine, proline, lysine, alanine, polyvinyl pyrrolidine,polyvinyl alcohol, polydextran, maltodextrins,hydroxypropyl-beta-cyclodextrin, partially hydrolysed starches, Tween-20or Tween-80.

In another embodiment of the present invention, a method for thepreparation of a long-term, storage stable adenovirus formulation,comprising the steps of providing adenovirus and combining theadenovirus with a solution comprising a buffer, a bulking agent, acryoprotectant and a lyoprotectant; and lyophilizing the solution,whereby lyophilization of the solution produces a freeze-dried cake ofthe adenovirus formulation that retains high infectivity and lowresidual moisture.

In particular embodiments, the bulking agent used for preparing thefreeze dried adenovirus formulation is mannitol, inositol, lactitol,xylitol, isomaltol, sorbitol, gelatin, agar, pectin, casein, dried skimmilk, dried whole milk, silicate, carboxypolymethylene, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose ormethylcellulose. In preferred embodiments, the bulking agent ismannitol, wherein mannitol comprises about 0.5% to about 8% (w/v) of theformulation.

In other embodiments, the cryoprotectant used for preparing the freezedried adenovirus formulation is sucrose, dextrose, lactose, trehalose,glucose, maltose, niacinamide, creatinine, monosodium glutamate dimethylsulfoxide or sweet whey solids. In preferred embodiments, thecryoprotectant is sucrose, wherein sucrose comprises about 2.5% to about10% (w/v) of said formulation.

In further embodiments, the lyoprotectant used for preparing the freezedried adenovirus formulation is human serum albumin, bovine serumalbumin, PEG, glycine, arginine, proline, lysine, alanine, polyvinylpyrrolidine, polyvinyl alcohol, polydextran, maltodextrins,hydroxypropyl-beta-cyclodextrin, partially hydrolysed starches, Tween-20or Tween-80. In preferred embodiments, the lyoprotectant is human serumalbumin.

In other embodiments, the buffer used for preparing the freeze driedadenovirus formulation is Tris-HCl, TES, HEPES, mono-Tris, brucinetetrahydrate, EPPS, tricine, or histidine, wherein the buffer is presentin the formulation at a concentration at about 1 mM to 50 mM. In onepreferred embodiment, the buffer used for preparing the freeze driedadenovirus formulation is Tris-HCl, wherein the Tris-HCl is included ina concentration of from about 1 mM to about 50 mM. In anotherembodiment, the Tris-HCl is included in a concentration of from about 5mM to about 20 mM. In still other embodiments, the freeze driedadenovirus formulation further comprises a salt selected from the groupconsisting of MgCl₂, MnCl₂, CaCl₂, ZnCl₂, NaCl and KCl.

In one embodiment, lyophilizing the adenovirus formulation is carriedout in the presence of an inert gas.

In certain embodiments, the method for preparing the freeze driedadenovirus formulation, wherein lyophilizing the solution comprises thesteps of, freezing the solution, subjecting the solution to a vacuum andsubjecting the solution to at least a first and a second drying cycle,whereby the second drying cycle reduces the residual moisture content ofthe freeze-dried cake to less than about 2%.

In another embodiment, a method for the preparation of a long-termstorage, stable adenovirus liquid formulation, comprising the steps ofproviding adenovirus and combining the adenovirus with a solutioncomprising a buffer and a polyol, whereby the adenovirus liquidformulation retains high infectivity.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various modifications withinthe spirit and scope of the invention will become apparent to thoseskilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Lyophilization Cycle of Adenovirus.

FIG. 2. Residual Moisture of Lyophilized Adenovirus After SecondaryDrying at 10° C.

FIG. 3. Stability of Lyophilized Adenovirus after Secondary Drying at110° C.

FIG. 4. Residual Moisture of Lyophilized Adenovirus After SecondaryDrying at 30° C.

FIG. 5. Stability of Lyophilized Adenovirus after Secondary Drying at30° C.

FIG. 6. HPLC Analysis of Lyophilized Adenovirus Stored at RoomTemperature.

FIG. 7. HPLC Analysis of Lyophilized Adenovirus Stored at 4° C.

FIG. 8. HPLC Analysis of Lyophilized Adenovirus Stored at −20° C.

FIG. 9A and FIG. 9B. Addition of DMSO to the formulation for anadenoviral vector increases the transduction efficiency. Human NSCLCxenografts were established on the flanks of nude mice. Animals receivedintratumoral injection of 2×10¹⁰ viral particles (vp) of Ad-βgalformulated in either PBS+glycerol (FIG. 9A and FIG. 9B, top panels) orin PBS+glycerol+5% DMSO (FIG. 9A and FIG. 9B, lower panels). Tumors wereexcised at either 24 (FIG. 9A) or 48 hours (FIG. 9B) post-injection andsectioned for histochemical analysis of reporter gene expression.Histochemical analysis was done on multiple sections from the tumorblock to analyze vector transduction and distribution. Two sections foreach formulation are illustrated: one from the tumor periphery (FIG. 9Aand FIG. 9B, left panels) and one from the center of the tumor (FIG. 9Aand FIG. 9B, right panels). In each section both transduction (asindicated by intensity of blue staining) and distribution (as indicatedby extent of blue staining) were improved by addition of DMSO to theformulation.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The need for long-term stable virus formulations that can be stored ator above refrigerated temperatures without losing infectivity is highlydesirable. Traditional methods of ultra-low temperature storage (≦60°C.) of virus preparations often limit the storage, transportation andclinical applications of viruses. The inventors have developed optimallyophilization formulations for freeze-drying adenovirus in which thefreeze-dried products maintain their stability (i.e., infectivity of60-100% of the starting infectivity) and have a residual moisture ofless than about 5% when stored for 6 months at 4° C.

In another embodiment, the inventors have developed long-term stableadenovirus formulations for storing adenovirus at 4° C. in a liquid formthat maintains stability (i.e., infectivity of 60-100% of the startinginfectivity) for at least 6 months.

A. PURIFICATION TECHNIQUES

A large scale process for the production and purification of adenovirusis described in U.S. Ser. No. 08/975,519 filed Nov. 20, 1997(specifically incorporated herein by reference without disclaimer). Thisproduction process offers not only scalability and validatability butalso virus purity comparable to that achieved using CsCl gradientultracentrifugation. This process involves the preparation ofrecombinant adenovirus particles, the process comprising preparing aculture of producer cells by seeding producer cells into a culturemedium, infecting cells in the culture after mid-log phase growth with arecombinant adenovirus comprising a selected recombinant gene operablylinked to a promoter, harvesting recombinant adenovirus particles fromthe cell culture and removing contaminating nucleic acids. An importantaspect of this process is the removal of contaminating nucleic acidsusing nucleases. Exemplary nucleases include Benzonase®, Pulmozyme®; orany other DNase or RNase commonly used within the art.

Enzymes such as Benzonase® degrade nucleic acid and have no proteolyticactivity. The ability of Benzonase® to rapidly hydrolyze nucleic acidsmakes the enzyme ideal for reducing cell lysate viscosity. It is wellknown that nucleic acids may adhere to cell derived particles such asviruses. The adhesion may interfere with separation due toagglomeration, change in size of the particle or change in particlecharge, resulting in little if any product being recovered with a givenpurification scheme. Benzonase® is well suited for reducing the nucleicacid load during purification, thus eliminating the interference andimproving yield.

As with all endonuclease, Benzonase® hydrolyzes internal phosphodiesterbonds between specific nucleotides. Upon complete digestion, all freenucleic acids present in solution are reduced to oligonucleotides 2 to 4bases in length.

The present invention further employs a number of different purificationtechniques to purify viral vectors of the present invention. Suchtechniques include those based on sedimentation and chromatography andare described in more detail herein below.

1. Density Gradient Centrifugation

There are two methods of density gradient centrifugation, the rate zonaltechnique and the isopycnic (equal density) technique, and both can beused when the quantitative separation of all the components of a mixtureof particles is required. They are also used for the determination ofbuoyant densities and for the estimation of sedimentation coefficients.

Particle separation by the rate zonal technique is based upondifferences in size or sedimentation rates. The technique involvescarefully layering a sample solution on top of a performed liquiddensity gradient, the highest density of which exceeds that of thedensest particles to be separated. The sample is then centrifuged untilthe desired degree of separation is effected, i.e., for sufficient timefor the particles to travel through the gradient to form discrete zonesor bands which are spaced according to the relative velocities of theparticles. Since the technique is time dependent, centrifugation must beterminated before any of the separated zones pellet at the bottom of thetube. The method has been used for the separation of enzymes, hormones,RNA-DNA hybrids, ribosomal subunits, subcellular organelles, for theanalysis of size distribution of samples of polysomes and forlipoprotein fractionations.

The sample is layered on top of a continuous density gradient whichspans the whole range of the particle densities which are to beseparated. The maximum density of the gradient, therefore, must alwaysexceed the density of the most dense particle. During centrifugation,sedimentation of the particles occurs until the buoyant density of theparticle and the density of the gradient are equal (i.e., wherep_(p)=p_(m) in equation 2.12). At this point no further sedimentationoccurs, irrespective of how long centrifugation continues, because theparticles are floating on a cushion of material that has a densitygreater than their own.

Isopycnic centrifugation, in contrast to the rate zonal technique, is anequilibrium method, the particles banding to form zones each at theirown characteristic buoyant density. In cases where, perhaps, not all thecomponents in a mixture of particles are required, a gradient range canbe selected in which unwanted components of the mixture will sediment tothe bottom of the centrifuge tube whilst the particles of interestsediment to their respective isopycnic positions. Such a techniqueinvolves a combination of both the rate zonal and isopycnic approaches.

Isopycnic centrifugation depends solely upon the buoyant density of theparticle and not its shape or size and is independent of time. Hencesoluble proteins, which have a very similar density (e.g., p=1.3 g cm⁻³in sucrose solution), cannot usually be separated by this method,whereas subcellular organelles (e.g., Golgi apparatus, p=1.11 g cm⁻³,mitochondria, p=1.19 g cm⁻³ and peroxisomes, p=1.23 g cm⁻³ in sucrosesolution) can be effectively separated.

As an alternative to layering the particle mixture to be separated ontoa preformed gradient, the sample is initially mixed with the gradientmedium to give a solution of uniform density, the gradient‘self-forming’, by sedimentation equilibrium, during centrifugation. Inthis method (referred to as the equilibrium isodensity method), use isgenerally made of the salts of heavy metals (e.g., cesium or rubidium),sucrose, colloidal silica or Metrizamide.

The sample (e.g., DNA) is mixed homogeneously with, for example, aconcentrated solution of cesium chloride. Centrifugation of theconcentrated cesium chloride solution results in the sedimentation ofthe CsCl molecules to form a concentration gradient and hence a densitygradient. The sample molecules (DNA), which were initially uniformlydistributed throughout the tube now either rise or sediment until theyreach a region where the solution density is equal to their own buoyantdensity, i.e. their isopycnic position, where they will band to formzones. This technique suffers from the disadvantage that often very longcentrifugation times (e.g., 36 to 48 hours) are required to establishequilibrium. However, it is commonly used in analytical centrifugationto determine the buoyant density of a particle, the base composition ofdouble stranded DNA and to separate linear from circular forms of DNA.

Many of the separations can be improved by increasing the densitydifferences between the different forms of DNA by the incorporation ofheavy isotopes (e.g., ¹⁵N) during biosynthesis, a technique used byLeselson and Stahl to elucidate the mechanism of DNA replication inEsherichia coli, or by the binding of heavy metal ions or dyes such asethidium bromide. Isopycnic gradients have also been used to separateand purify viruses and analyze human plasma lipoproteins.

2. Chromatography

Purification techniques are well known to those of skill in the art.These techniques tend to involve the fractionation of the cellularmilieu (e.g., density gradient centrifugation) to separate theadenovirus particles from other components of the mixture. Havingseparated adenoviral particles from the other components, the adenovirusmay be purified using chromatographic and electrophoretic techniques toachieve complete purification. Analytical methods particularly suited tothe preparation of a pure adenoviral particle of the present inventionare ion-exchange chromatography, size exclusion chromatography andpolyacrylamide gel electrophoresis. A particularly efficientpurification method to be employed in conjunction with the presentinvention is HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of anadenoviral particle. The term “purified” as used herein, is intended torefer to a composition, isolatable from other components, wherein theadenoviral particle is purified to any degree relative to itsnaturally-obtainable form. A purified adenoviral particle therefore alsorefers to an adenoviral component, free from the environment in which itmay naturally occur.

Generally, “purified” will refer to an adenoviral particle that has beensubjected to fractionation to remove various other components, and whichcomposition substantially retains its expressed biological activity.Where the term “substantially purified” is used, this designation willrefer to a composition in which the particle, protein or peptide formsthe major component of the composition, such as constituting about 50%or more of the constituents in the composition.

Various methods for quantifying the degree of purification of a proteinor peptide will be known to those of skill in the art in light of thepresent disclosure. These include, for example, determining the specificactivity of an active fraction, or assessing the amount of polypeptideswithin a fraction by SDS/PAGE analysis. A preferred method for assessingthe purity of a fraction is to calculate the specific activity of thefraction, to compare it to the specific activity of the initial extract,and to thus calculate the degree of purity, herein assessed by a “-foldpurification number”. The actual units used to represent the amount ofactivity will, of course, be dependent upon the particular assaytechnique chosen to follow the purification and whether or not theexpressed protein or peptide exhibits a detectable activity.

There is no general requirement that the adenovirus, always be providedin their most purified state. Indeed, it is contemplated that lesssubstantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater-fold purification than thesame technique utilizing a low pressure chromatography system. Methodsexhibiting a lower degree of relative purification may have advantagesin total recovery of protein product, or in maintaining the activity ofan expressed protein.

Of course, it is understood that the chromatographic techniques andother purification techniques known to those of skill in the art mayalso be employed to purify proteins expressed by the adenoviral vectorsof the present invention. Ion exchange chromatography and highperformance liquid chromatography are exemplary purification techniquesemployed in the purification of adenoviral particles and are describedin further detail herein below.

a. Ion-Exchange Chromatography

The basic principle of ion-exchange chromatography is that the affinityof a substance for the exchanger depends on both the electricalproperties of the material and the relative affinity of other chargedsubstances in the solvent. Hence, bound material can be eluted bychanging the pH, thus altering the charge of the material, or by addingcompeting materials, of which salts are but one example. Becausedifferent substances have different electrical properties, theconditions for release vary with each bound molecular species. Ingeneral, to get good separation, the methods of choice are eithercontinuous ionic strength gradient elution or stepwise elution. (Agradient of pH alone is not often used because it is difficult to set upa pH gradient without simultaneously increasing ionic strength.) For ananion exchanger, either pH and ionic strength are gradually increased orionic strength alone is increased. For a cation exchanger, both pH andionic strength are increased. The actual choice of the elution procedureis usually a result of trial and error and of considerations ofstability. For example, for unstable materials, it is best to maintainfairly constant pH.

An ion exchanger is a solid that has chemically bound charged groups towhich ions are electrostatically bound; it can exchange these ions forions in aqueous solution. Ion exchangers can be used in columnchromatography to separate molecules according to charge; actually otherfeatures of the molecule are usually important so that thechromatographic behavior is sensitive to the charge density, chargedistribution, and the size of the molecule.

The principle of ion-exchange chromatography is that charged moleculesadsorb to ion exchangers reversibly so that molecules can be bound oreluted by changing the ionic environment. Separation on ion exchangersis usually accomplished in two stages: first, the substances to beseparated are bound to the exchanger, using conditions that give stableand tight binding; then the column is eluted with buffers of differentpH, ionic strength, or composition and the components of the buffercompete with the bound material for the binding sites.

An ion exchanger is usually a three-dimensional network or matrix thatcontains covalently linked charged groups. If a group is negativelycharged, it will exchange positive ions and is a cation exchanger. Atypical group used in cation exchangers is the sulfonic group, SO₃ ⁻. Ifan H⁺ is bound to the group, the exchanger is said to be in the acidform; it can, for example, exchange on H⁺ for one Na⁺ or two H⁺ for oneCa²⁺. The sulfonic acid group is called a strongly acidic cationexchanger. Other commonly used groups are phenolic hydroxyl andcarboxyl, both weakly acidic cation exchangers. If the charged group ispositive—for example, a quaternary amino group—it is a strongly basicanion exchanger. The most common weakly basic anion exchangers arearomatic or aliphatic amino groups.

The matrix can be made of various material. Commonly used materials aredextran, cellulose, agarose and copolymers of styrene and vinylbenzenein which the divinylbenzene both cross-links the polystyrene strands andcontains the charged groups. Table 1 gives the composition of many ionexchangers.

The total capacity of an ion exchanger measures its ability to take upexchangeable groups per milligram of dry weight. This number is suppliedby the manufacturer and is important because, if the capacity isexceeded, ions will pass through the column without binding.

TABLE 1 Matrix Exchanger Functional Group Tradename Dextran StrongCationic Sulfopropyl SP-Sephadex Weak Cationic Carboxymethyl CM-SephadexStrong Anionic Diethyl-(2- QAE-Sephadex hydroxypropyl)- aminoethyl WeakAnionic Diethylaminoethyl DEAE-Sephadex Cellulose Cationic CarboxymethylCM-Cellulose Cationic Phospho P-cel Anionic DiethylaminoethylDEAE-cellulose Anionic Polyethylenimine PEI-Cellulose AnionicBenzoylated- DEAE(BND)- naphthoylated, cellulose deiethylaminoethylAnionic p-Aminobenzyl PAB-cellulose Styrene- Strong Cationic Sulfonicacid AG 50 divinyl- Strong Anionic AG 1 benzene Strong Sulfonic acid +AG 501 Cationic + Tetra- Strong Anionic methylammonium Acrylic WeakCationic Carboxylic Bio-Rex 70 Phenolic Strong Cationic Sulfonic acidBio-Rex 40 Expoxyamine Weak Anionic Tertiary amino AG-3

The available capacity is the capacity under particular experimentalconditions (i.e., pH, ionic strength). For example, the extent to whichan ion exchanger is charged depends on the pH (the effect of pH issmaller with strong ion exchangers). Another factor is ionic strengthbecause small ions near the charged groups compete with the samplemolecule for these groups. This competition is quite effective if thesample is a macromolecule because the higher diffusion coefficient ofthe small ion means a greater number of encounters. Clearly, as bufferconcentration increases, competition becomes keener.

The porosity of the matrix is an important feature because the chargedgroups are both inside and outside the matrix and because the matrixalso acts as a molecular sieve. Large molecules may be unable topenetrate the pores; so the capacity will decease with increasingmolecular dimensions. The porosity of the polystyrene-based resins isdetermined by the amount of cross-linking by the divinylbenzene(porosity decreases with increasing amounts of divinylbenzene). With theDowex and AG series, the percentage of divinylbenzene is indicated by anumber after an X—hence, Dowex 50-X8 is 8% divinylbenzene

Ion exchangers come in a variety of particle sizes, called mesh size.Finer mesh means an increased surface-to-volume ration and thereforeincreased capacity and decreased time for exchange to occur for a givenvolume of the exchanger. On the other hand, fine mesh means a slow flowrate, which can increase diffusional spreading. The use of very fineparticles, approximately 10 μm in diameter and high pressure to maintainan adequate flow is called high-performance or high-pressure liquidchromatography or simply HPLC.

Such a collection of exchangers having such different properties—charge,capacity, porosity, mesh—makes the selection of the appropriate one foraccomplishing a particular separation difficult. How to decide on thetype of column material and the conditions for binding and elution isdescribed in the following Examples.

There are a number of choice to be made when employing ion exchangechromatography as a technique. The first choice to be made is whetherthe exchanger is to be anionic or cationic. If the materials to be boundto the column have a single charge (i.e., either plus or minus), thechoice is clear. However, many substances (e.g., proteins, viruses),carry both negative and positive charges and the net charge depends onthe pH. In such cases, the primary factor is the stability of thesubstance at various pH values. Most proteins have a pH range ofstability (i.e., in which they do not denature) in which they are eitherpositively or negatively charged. Hence, if a protein is stable at pHvalues above the isoelectric point, an anion exchanger should be used;if stable at values below the isoelectric point, a cation exchanger isrequired.

The choice between strong and weak exchangers is also based on theeffect of pH on charge and stability. For example, if a weakly ionizedsubstance that requires very low or high pH for ionization ischromatographed, a strong ion exchanger is called for because itfunctions over the entire pH range. However, if the substance is labile,weak ion exchangers are preferable because strong exchangers are oftencapable of distorting a molecule so much that the molecule denatures.The pH at which the substance is stable must, of course, be matched tothe narrow range of pH in which a particular weak exchanger is charged.Weak ion exchangers are also excellent for the separation of moleculeswith a high charge from those with a small charge, because the weaklycharged ions usually fail to bind. Weak exchangers also show greaterresolution of substances if charge differences are very small. If amacromolecule has a very strong charge, it may be impossible to elutefrom a strong exchanger and a weak exchanger again may be preferable. Ingeneral, weak exchangers are more useful than strong exchangers.

The Sephadex and Bio-gel exchangers offer a particular advantage formacromolecules that are unstable in low ionic strength. Because thecross-links in these materials maintain the insolubility of the matrixeven if the matrix is highly polar, the density of ionizable groups canbe made several times greater than is possible with cellulose ionexchangers. The increased charge density means increased affinity sothat adsorption can be carried out at higher ionic strengths. On theother hand, these exchangers retain some of their molecular sievingproperties so that sometimes molecular weight differences annul thedistribution caused by the charge differences; the molecular sievingeffect may also enhance the separation.

Small molecules are best separated on matrices with small pore size(high degree of cross-linking) because the available capacity is large,whereas macromolecules need large pore size. However, except for theSephadex type, most ion exchangers do not afford the opportunity formatching the porosity with the molecular weight.

The cellulose ion exchangers have proved to be the best for purifyinglarge molecules such as proteins and polynucleotides. This is becausethe matrix is fibrous, and hence all functional groups are on thesurface and available to even the largest molecules. In may caseshowever, beaded forms such as DEAE-Sephacel and DEAE-Biogel P are moreuseful because there is a better flow rate and the molecular sievingeffect aids in separation.

Selecting a mesh size is always difficult. Small mesh size improvesresolution but decreases flow rate, which increases zone spreading anddecreases resolution. Hence, the appropriate mesh size is usuallydetermined empirically.

Because buffers themselves consist of ions, they can also exchange, andthe pH equilibrium can be affected. To avoid these problems, the rule ofbuffers is adopted: use cationic buffers with anion exchangers andanionic buffers with cation exchangers. Because ionic strength is afactor in binding, a buffer should be chosen that has a high bufferingcapacity so that its ionic strength need not be too high. Furthermore,for best resolution, it has been generally found that the ionicconditions used to apply the sample to the column (the so-calledstarting conditions) should be near those used for eluting the column.

b. High Performance Liquid Chromatography

High performance liquid chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

B. VIRAL FORMULATION

Retrovirus, adenovirus, adeno-associated virus, and herpes simplex virusare the most commonly used viruses in gene therapy (Robbins andGhivizzani; 1998). It is contemplated in the present invention that thepreparation of long-term stable adenovirus vectors that can be stored ator above refrigerated temperatures would be useful as gene therapyvectors. Viral particles must maintain their structural integrity toremain infective and biologically active for use as gene therapyvectors. Current virus formulations do not readily make it feasible tostore or transport viral vector at or above refrigerated temperatureswithout significant loss of viral infectivity.

The present invention describes long-term stable adenovirus formulationsthat can be stored at 4° C. for periods up to 6 months. In oneembodiment of the present invention, adenovirus preparations areformulated for lyophilization and long-term storage at 4° C. asfreeze-dried adenovirus. In another embodiment, the adenovirus isprepared as a liquid formulation that is long-term stable at 4° C. Animportant aspect of both the lyophilized and liquid adenovirusformulations is the addition of at least one or more compounds thatimprove the long-term, storage stability of the adenovirus.

The term “compound” in the context of the present invention includespharmaceutically acceptable carriers such as bulking agents,cryoprotectants, lyoprotectants, preservatives, solvents, solutes andany additional pharmaceutical agents well known in the art. Bufferingagents and other types of pH control can also be added simultaneously inorder to provide for maximum buffering capacity for the adenovirusformulation. For example, pH changes that deviate from physiologicalconditions often result in irreversible aggregation of proteins (Wetzel,1992) and viral capsids (Misselwitz et al., 1995) due to complete orpartial denaturation of the protein. Thus, buffering agents areparticularly important for virus preparations that aggregate or denatureat sub-optimal pH ranges.

1. Lyophilized Formulations

The formulation of lyophilized, long-term storage stable adenovirus inthe present invention requires the presence of one or more excipients.More particularly, for optimal long-term stability of lyophilizedadenovirus formulations, a bulking agent and one or more protectants aredesirable. It is well known in the art that loss in virus infectivityoften is directly related to denaturation, self association andaggregation of the viral particles (Misselwitz et al., 1995;Vanlandschoot et al., 1998; Sagrera et al., 1998; Lu et al., 1998). Infact, the E. coli heat shock proteins GroEL/GroES have been shown toboth stabilize viral particles from denaturation and aggregation duringhigh stress cellular conditions and to facilitate capsid assembly duringnon-stressed, normal cellular conditions (Polissi et al., 1995;Nakonechny and Teschke, 1998).

The use of bulking agents, cryoprotectants, lyoprotectants and salts inthe present invention are included in the formulation of lyophilizedadenovirus to improve long-term stability (i.e. infectivity) of theadenovirus freeze-dried products. The stabilizing effect of thecryoprotectant sucrose against irreversible denaturation and aggregationhas been described previously as an excluded volume effect (Hall et al.,1995). Similarly, bulking agents, cryo- and lyoprotectants such aspolyacrylamide gels, agaorse gels, dextran and polyethylene glycol (PEG)have demonstrated enhanced stabilities of proteins and nucleic acids inpart by excluded volume effects (Fried and Bromberg, 1997; Vossen andFried, 1997). The exact mechanistic details of excluded volume effectsare still not clear. A currently accepted theory is that many of thesecompounds result in the preferential hydration of protein molecules(i.e. volume of exclusion), which tends to stabilize the native versusthe denatured conformation of proteins, and therefore preventsaggregation. In addition, the presence of low concentrations ofcosolvents (e.g., salts) result in charge screening of proteins andviral protein coats increasing their solubility in water.

The use of bulking agents, cryoprotectants, lyoprotectants and salts inthe present invention are contemplated and demonstrated experimentallyto improve the storage stability of lyophilized adenovirus products. Inone embodiment, a bulking agent and protectants are combined with abuffer comprising adenovirus.

Bulking agents, cryoprotectants and lyoprotectants are well known in theart (Lueckel et al., 1998; Herman et al., 1994; Croyle et al., 1998;Corveleyn and Remon, 1996). Bulking agents considered in the presentinvention are mannitol, inositol, lactitol, xylitol, isomaltol,sorbitol, gelatin, agar, pectin, casein, dried skim milk, dried wholemilk, silicate, carboxypolymethylene, hydroxyethyl cellulose,hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcelluloseand other bulking agents well known in the art. Cryoprotectantsconsidered are sucrose, dextrose, lactose, trehalose, glucose, maltose,niacinamide, creatinine, monosodium glutamate, dimethyl sulfoxide, sweetwhey solids, as well as other known cryoprotectants. Lyoprotectantscontemplated for use in the present invention are human serum albumin,bovine serum albumin, PEG, glycine, arginine, proline, lysine, alanine,polyvinyl pyrrolidine, polyvinyl alcohol, polydextran, maltodextrins,hydroxypropyl-beta-cyclodextrin, partially hydrolysed starches, Tween-20and Tween-80. Certain lyoprotectants are also classified ascryoprotectants and vice versa. For the purpose of the presentinvention, cryoprotectants and lyoprotectants are represented asindependent classes of compounds. However, this classification is onlyfor clarity of the invention and should not limit the person skilled inthe art from using any excipient that stabilizes the adenovirusformulation. In other embodiments of the present invention, the termexcipient encompasses bulking agents, cryo- and lyoprotectants. Incertain embodiments, salts are included in the formulation in additionto the aforementioned excipients. The following salts are considered foruse in the present invention MgCl₂, MnCl₂, CaCl₂, ZnCl₂, NaCl, and KCl,but should not preclude the use of other salts that improve stability ofthe adenovirus formulation.

In other embodiments of the invention, the lyophilized adenovirusformulation is dried in the presence of an inert gas or a combination ofinert gasses. The purging of the lyophilization vessel with an inert gasor gasses, the presence of the inert gas or gasses during lyophilizationof the adenovirus solution and during the capping of the lyophilizationvial after the drying step, are contemplated to minimize the deleteriouseffects of O₂. It is known that residual O₂ leads to oxidation anddegradation of proteins. It is contemplated that purging and capping ofthe freeze-dried adenovirus product improves the long-term storagestability of the adenovirus product. The use of antioxidants such asβ-mercapto ethanol, DTT, citric acid and the like may also be consideredfor use in formulations.

An important aspect of the lyophilization process is a second dryingcycle. The second drying cycle is at a temperature of 30° C. for atleast 3.5 hours, which is demonstrated to reduce the residual moistureof the adenovirus freeze-dried product to less than 2% water immediatelyafter drying. It is contemplated that the reduced residual moistureimproves the long-term storage stability of the adenovirus freeze-driedproduct. Longer drying times up to 20 hours are thus contemplated tofurther reduce residual moisture.

2. Liquid Formulations

The formulation of liquid, long-term storage stable adenovirus in thepresent invention requires the presence of a polyol. A polyol is apolyhydric alcohol containing two or more hydroxyl groups. For optimallong-term stability of liquid adenovirus formulations in the presentinvention, glycerol is used. In particular embodiments of the invention,the presence 20% glycerol results in adenovirus stability (80% PFU/mL)for periods of time at least up to 6 months days when stored at 4° C.

Glycerol (glycerin) is one of the oldest and most widely used excipientsin pharmaceutical products. It is a clear, colorless liquid which ismiscible with water and alcohol. Glycerol is hygroscopic, stable to mildacidic and basic environments and can be sterilized at temperatures upto 150° C. It is well known as both a taste masking and cryoprotectiveagent, as well as an antimicrobial agent. It has good solubilizing powerand is a commonly used solvent in parenteral formulations. It isconsidered to be one of the safest excipients used since it ismetabolized to glucose, or to substances which are involved withtriglyceride synthesis or glycolysis (Frank et al., 1981). It is a GRASlisted excipient and typically used at levels up to 50% in parenteralformulations.

The stabilizing effects of glycerol on protein structure is well knownin the art (Hase et al., 1998; Juranville et al., 1998). Several studiesindicate that glycerol has a similar effect of viral particles. Forexample, when competent Haemophilus influenza bacteria were exposed topurified phage and plated for transfectants, a 100-fold increase intransfectants was observed when 32% glycerol was present in the solution(Stuy, 1986) In yet another study, glycerol was demonstrated to preservethe integrity of vaccinia virus (Slonin and Roslerova, 1969).

Other polyols contemplated for use in the present invention arepolyethylene glycol, propylene glycol, sorbitol, mannitol, and the like.Polyethylene glycols are polymers of ethylene oxide with the generalformula:HO—CH₂—(CH₂—O—CH₂)_(n)—CH₂OH

where n represents the number of oxyethylene groups. The PEG's aredesignated by a numerical value, which is indicative of the averagemolecular weight for a given grade. Molecular weights below 600 areliquids, and molecular weights above 1000 are solids at roomtemperature. These polymers are readily soluble in water, which makethem quite useful for parenteral dosage forms. Only PEG 400 and PEG 300are utilized in parenteral products, typically at concentrations up to30% v/v. These polymers are generally regarded as non-toxic andnon-irritating. There are numerous reviews regarding the pharmaceuticaland toxicological characteristics of these polyols (Smyth et al., 1950;Rowe and Wolf, 1982, Swarbrick and Boylan, 1990).

Propylene glycol, a dihydroxy alcohol, is one of the more commonsolvents encountered in pharmaceutical cosolvent formulations, for bothparenteral and non-parenteral dosage forms. PG is generally regarded asnon-toxic. It is more hygroscopic than glycerin, and has excellentsolubilizing power for a wide variety of compounds. In addition, it hasexcellent bacteriocidal and preservative properties (Heine et al.,1950). PG is metabolized to carbon dioxide and water via lactic andpyruvic acid intermediates and, therefore, not prone to the severetoxicities.

Sorbitol and mannitol are hexahydric alcohols, consisting of white,crystalline powders, that are soluble in water. Both are commonly usedexcipients in pharmaceutical products with little or no toxicityassociated, as approved by the FDA for food use. The mechanistics ofsorbitol and mannitol protein and viral stabilization is still notcompletely understood. Current theories suggest at least part of theeffect is osmotic diuretic (Vanholder, et al., 1984; de Rizzo, et al.,1988). The use of the polyols described above are considered exemplary,but should not limit the skilled artist from selecting other polyolsthat confer viral stability for liquid formulations.

It is also contemplated, in addition to a polyol in the liquidformulation, that one or possibly two excipients may also be included.Excipients considered for use in the present invention are inositol,lactitol, xylitol, isomaltol, gelatin, agar, pectin, casein, dried skimmilk, dried whole milk, silicate, carboxypolymethylene, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose, sucrose, dextrose, lactose, trehalose, glucose,maltose, niacinamide, creatinine, monosodium glutamate, dimethylsulfoxide, sweet whey solids, human serum albumin, bovine serum albumin,glycine, arginine, proline, lysine, alanine, polyvinyl pyrrolidine,polyvinyl alcohol, polydextran, maltodextrins,hydroxypropyl-beta-cyclodextrin, partially hydrolysed starches, Tween-20and Tween-80. The choice of a particular excipient is dependent in someinstances on the desired properties of the viral formulation.

In particular embodiments, dimethyl sulfoxide (DMSO) is contemplated foruse in the present invention. DMSO has been demonstrated to enhance theinfectivity of adenovirus preparations by increasing the efficiency ofgene transfer (Chikada and Jones, 1999). For example, the infectivity ofadenovirus type 2 DNA in 293 cells was increased up to five-fold by thebrief treatment of cell monolayers with 25% DMSO (Chinnadurai et al.,1978) The stabilization of virus particles via DMSO also has beenreported (Wallis and Melnick, 1968). The present inventors demonstratethat the intratumoral administration of Ad-p53 is improved when DMSO isadded to 5 or 10% (see FIG. 9). Adenovirus studies via intravesicaladministration indicate that an adenoviral vector may be stable in up to50% DMSO (WO 98/35554). In other embodiments, a polyol contemplated foruse in the present invention as an enhancer of adenovirus genetransduction is a polyoxyalkene (U.S. Pat. No. 5,552,309, specificallyincorporate herein by reference in its entirety).

Thus in particular embodiments, an adenoviral formulation according tothe present invention may also contain DMSO. The concentration forintratumoral administration may contain from about 2% to 67% DMSO,preferably from about 5% to 20%. The concentration for intravesicaladministration may contain from about 2% to 67% DMSO, preferably fromabout 20% to 50%. The concentration for topical administration maycontain from about 2% to 67% DMSO, preferably from about 10% to 40%. Theconcentration for intra-articular administration may contain from about2% to 67% DMSO, preferably, from about 5% to 40%. The concentration forsystemic administration may contain from about 2% to 75% DMSO,preferably from about 50% to 67%.

Adenovirus polyol formulations of the invention may future comprise apolyoxamer, such as Polyoxamer 407, at concentrations of from about 0.5%to 20%, preferably from about 10% to 20%. The formulation storage stableadenovirus may also contain from about 5% to 40% dimethylacetamide,preferably from about 10% to 25%, Or it may contain from about 10% to50% of a polyethylene glycol, such as polyethylene glycol 400,preferably from about 15% to 50%. Of course, the formulation of saidadenovirus also may contain combinations of the above components.

C. VIRAL TRANSFORMATION

The present invention employs, in one example, adenoviral infection ofcells in order to generate therapeutically significant vectors.Typically, the virus will simply be exposed to the appropriate host cellunder physiologic conditions, permitting uptake of the virus. Thoughadenovirus is exemplified, the present methods may be advantageouslyemployed with other viral vectors, as discussed below.

1. Viral Infection

a. Adenovirus

One method for delivery of the recombinant DNA involves the use of anadenovirus expression vector. Although adenovirus vectors are known tohave a low capacity for integration into genomic DNA, this feature iscounterbalanced by the high efficiency of gene transfer afforded bythese vectors. “Adenovirus expression vector” is meant to include thoseconstructs containing adenovirus sequences sufficient to (a) supportpackaging of the construct and (b) to ultimately express a recombinantgene construct that has been cloned therein.

The vector comprises a genetically engineered form of adenovirus.Knowledge of the genetic organization or adenovirus, a 36 kb, linear,double-stranded DNA virus, allows substitution of large pieces ofadenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz,1992). In contrast to retrovirus, the adenoviral infection of host cellsdoes not result in chromosomal integration because adenoviral DNA canreplicate in an episomal manner without potential genotoxicity. Also,adenoviruses are structurally stable, and no genome rearrangement hasbeen detected after extensive amplification.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget-cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off (Renan,1990). The products of the late genes, including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (MLP).The MLP, (located at 16.8 m.u.) is particularly efficient during thelate phase of infection, and all the mRNA's issued from this promoterpossess a 5′-tripartite leader (TPL) sequence which makes them preferredmRNA's for translation.

In a current system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones and Shenk, 1978), the current adenovirus vectors, with the helpof 293 cells, carry foreign DNA in either the E1, the D3 or both regions(Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury et al.,1987), providing capacity for about 2 extra kb of DNA. Combined with theapproximately 5.5 kb of DNA that is replaceable in the E1 and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kb, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone.

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

Racher et al. (1995) have disclosed improved methods for culturing 293cells and propagating adenovirus. In one format, natural cell aggregatesare grown by inoculating individual cells into 1 liter siliconizedspinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium.Following stirring at 40 rpm, the cell viability is estimated withtrypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin,Stone, UK) (5 g/l) is employed as follows. A cell inoculum, resuspendedin 5 ml of medium, is added to the carrier (50 ml) in a 250 mlErlenmeyer flask and left stationary, with occasional agitation, for 1to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication defective and will not have an adenovirus E1 region.Thus, it will be most convenient to introduce the transforming constructat the position from which the E1-coding sequences have been removed.However, the position of insertion of the construct within theadenovirus sequences is not critical to the invention. Thepolynucleotide encoding the gene of interest may also be inserted inlieu of the deleted E3 region in E3 replacement vectors as described byKarlsson et al. (1986) or in the E4 region where a helper cell line orhelper virus complements the E4 defect.

Adenovirus growth and manipulation is known to those of skill in theart, and exhibits broad host range in vitro and in vivo. This group ofviruses can be obtained in high titers, e.g., 10⁹-10¹¹ plaque-formingunits per ml, and they are highly infective. The life cycle ofadenovirus does not require integration into the host cell genome. Theforeign genes delivered by adenovirus vectors are episomal and,therefore, have low genotoxicity to host cells. No side effects havebeen reported in studies of vaccination with wild-type adenovirus (Couchet al., 1963; Top et al., 1971), demonstrating their safety andtherapeutic potential as in vivo gene transfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1992). Adenoviral vectors alsohave been described for treatment of certain types of cancers (U.S. Pat.No. 5,789,244, specifically incorporated herein by reference in itsentirety). Animal studies have suggested that recombinant adenoviruscould be used for gene therapy (Stratford-Perricaudet and Perricaudet,1991; Stratford-Perricaudet et al., 1990; Rich et al., 1993). Studies inadministering recombinant adenovirus to different tissues includetrachea instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992),muscle injection (Ragot et al., 1993), peripheral intravenous injections(Herz and Gerard, 1993) and stereotactic inoculation into the brain (LeGal La Salle et al., 1993).

b. Retrovirus

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains three genes,gag, pol, and env that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene contains a signal for packaging of the genome into virions. Twolong terminal repeat (LTR) sequences are present at the 5′ and 3′ endsof the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding agene of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

Concern with the use of defective retrovirus vectors is the potentialappearance of wild-type replication-competent virus in the packagingcells. This can result from recombination events in which the intactsequence from the recombinant virus inserts upstream from the gag, pol,env sequence integrated in the host cell genome. However, new packagingcell lines are now available that should greatly decrease the likelihoodof recombination (Markowitz et al., 1988; Hersdorffer et al., 1990).

c. Adeno-Associated Virus

Adeno-associated virus (AAV) is an attractive vector system for use inthe present invention as it has a high frequency of integration and itcan infect nondividing cells, thus making it useful for delivery ofgenes into mammalian cells in tissue culture (Muzyczka, 1992). AAV has abroad host range for infectivity (Tratschin, et al., 1984; Laughlin, etal., 1986; Lebkowski, et al., 1988; McLaughlin, et al., 1988), whichmeans it is applicable for use with the present invention. Detailsconcerning the generation and use of rAAV vectors are described in U.S.Pat. No. 5,139,941 and U.S. Pat. No. 4,797,368, each incorporated hereinby reference.

Studies demonstrating the use of AAV in gene delivery include LaFace etal. (1988); Zhou et al. (1993); Flotte et al. (1993); and Walsh et al.(1994). Recombinant AAV vectors have been used successfully for in vitroand in vivo transduction of marker genes (Kaplitt et al., 1994;Lebkowski et al., 1988; Samulski et al., 1989; Shelling and Smith, 1994;Yoder et al., 1994; Zhou et al., 1994; Hermonat and Muzyczka, 1984;Tratschin et al., 1985; McLaughlin et al., 1988) and genes involved inhuman diseases (Flotte et al., 1992; Luo et al., 1994; Ohi et al., 1990;Walsh et al., 1994; Wei et al., 1994). Recently, an AAV vector has beenapproved for phase I human trials for the treatment of cystic fibrosis.

AAV is a dependent parvovirus in that it requires coinfection withanother virus (either adenovirus or a member of the herpes virus family)to undergo a productive infection in cultured cells (Muzyczka, 1992). Inthe absence of coinfection with helper virus, the wild-type AAV genomeintegrates through its ends into human chromosome 19 where it resides ina latent state as a provirus (Kotin et al., 1990; Samulski et al.,1991). rAAV, however, is not restricted to chromosome 19 for integrationunless the AAV Rep protein is also expressed (Shelling and Smith, 1994).When a cell carrying an AAV provirus is superinfected with a helpervirus, the AAV genome is “rescued” from the chromosome or from arecombinant plasmid, and a normal productive infection is established(Samulski et al., 1989; McLaughlin et al., 1988; Kotin et al., 1990;Muzyczka, 1992).

Typically, recombinant AAV (rAAV) virus is made by cotransfecting aplasmid containing the gene of interest flanked by the two AAV terminalrepeats (McLaughlin et al., 1988; Samulski et al., 1989; eachincorporated herein by reference) and an expression plasmid containingthe wild-type AAV coding sequences without the terminal repeats, forexample pIM45 (McCarty et al., 1991; incorporated herein by reference).The cells are also infected or transfected with adenovirus or plasmidscarrying the adenovirus genes required for AAV helper function. rAAVvirus stocks made in such fashion are contaminated with adenovirus whichmust be physically separated from the rAAV particles (for example, bycesium chloride density centrifugation). Alternatively, adenovirusvectors containing the AAV coding regions or cell lines containing theAAV coding regions and some or all of the adenovirus helper genes couldbe used (Yang et al., 1994a; Clark et al., 1995). Cell lines carryingthe rAAV DNA as an integrated provirus can also be used (Flotte et al.,1995).

d. Other Viral Vectors

Other viral vectors may be employed as constructs in the presentinvention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988) andherpesviruses may be employed. They offer several attractive featuresfor various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwaland Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. Chang et al. recently introduced thechloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virusgenome in the place of the polymerase, surface, and pre-surface codingsequences. It was cotransfected with wild-type virus into an avianhepatoma cell line. Culture media containing high titers of therecombinant virus were used to infect primary duckling hepatocytes.Stable CAT gene expression was detected for at least 24 days aftertransfection (Chang et al., 1991).

Also contemplated for use in the present invention is a fairly new classof viruses termed oncolytic virus (Pennisi, 1998). Some of the virusesincluded in this group are reovirus, the genetically modified adenovirusOYNX-015 and CN706. These oncolytic viruses, which have not beengenetically altered to prevent their replication, destroy certain typesof cancer cells by multiplying and spreading, killing only the cancercells. Each of the above oncolytic viruses are proposed to operate viadifferent pathways involved in cancers.

For example, human reovirus requires an activated Ras signaling pathwayfor infection of cultured cells. Thus, in certain tumors with anoveractive ras gene, reovirus readily replicates. In a study onreovirus, severe combined immune deficient mice bearing tumorsestablished from v-erbB-transformed murine NIH 3T3 cells or human U87glioblastoma cells were treated with the virus. A single intratumoralinjection of virus resulted in regression of tumors in 65% to 80% of themice. Treatment of immune-competent C3H mice bearing tumors establishedfrom a ras-transformed C3H-10T1/2 cells also resulted in tumorregression, although a series of injections were required (Coffey etal., 1998)

2. Vectors and Regulatory Signals

Vectors of the present invention are designed, primarily, to transformcells with a gene under the control of regulated eukaryotic promoters(i.e., inducible, repressable, tissue specific). Also, the vectorsusually will contain a selectable marker if, for no other reason, tofacilitate their production in vitro. However, selectable markers mayplay an important role in producing recombinant cells and thus adiscussion of promoters is useful here. Table 2 and Table 3 below, listinducible promoter elements and enhancer elements, respectively.

TABLE 2 Inducible Elements Element Inducer References MT II PhorbolEster (TFA) Palmiter et al., 1982; Haslinger and Heavy metals Karin,1985; Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987;Karin ®, 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV (mouseGlucocorticoids Huang et al., 1981; Lee et al., 1981; mammary tumorvirus) Majors and Varmus, 1983; Chandler et al., 1983; Lee et al., 1984;Fonta et al., 1985; Sakai et al., 1986 β-Interferon poly(rI) × polyTavernier et al., 1983 (rc) Adenovirus 5 E2 Ela Imperiale and Nevins,1984 Collagenase Phorbol Ester (TPA) Angle et al., 1987a StromelysinPhorbol Ester (TPA) Angle et al., 1987b SV40 Phorbol Ester (TFA) Angelet al., 1987b Murine MX Gene Interferon, Newcastle Disease Virus GRP78Gene A23187 Resendez et al., 1988 α-2-Macroglobulin IL-6 Kunz et al.,1989 Vimentin Serum Rittling et al., 1989 MHC Class I Gene H-2κbInterferon Blanar et al., 1989 HSP70 Ela, SV40 Large T Taylor et al.,1989; Taylor and Antigen Kingston, 1990a, b Proliferin Phorbol Ester-TPAMordacq and Linzer, 1989 Tumor Necrosis Factor FMA Hensel et al., 1989Thyroid Stimulating Thyroid Hormone Chatterjee et al., 1989 Hormone αGene

TABLE 3 Other Promoter/Enhancer Elements Promoter/Enhancer ReferencesImmunoglobulin Heavy Chain Hanerji et al., 1983; Gilles et al., 1983;Grosschedl and Baltimore, 1985; Atchinson and Perry, 1986, 1987; Imleret al., 1987; Weinberger et al., 1988; Kiledjian et al., 1988; Porton etal., 1990 Immunoglobulin Light Chain Queen and Baltimore, 1983; Picardand Schaffner, 1984 T-Cell Receptor Luria et al., 1987, Winoto andBaltimore, 1989; Redondo et al., 1990 HLA DQ α and DQ β Sullivan andPeterlin, 1987 β-Interferon Goodbourn et al., 1986; Fujita et al., 1987;Goodbourn and Maniatis, 1985 Interleukin-2 Greene et al., 1989Interleukin-2 Receptor Greene et al., 1989; Lin et al., 1990 MHC ClassII 5 Koch et al., 1989 MHC Class II HLA-DRα Sherman et al., 1989 β-ActinKawamoto et al., 1988; Ng et al., 1989 Muscle Creatine Kinase Jaynes etal., 1988; Horlick and Benfield, 1989; Johnson et al., 1989a Prealbumin(Transthyretin) Costa et al., 1988 Elastase I Omitz et al., 1987Metallothionein Karin et al., 1987; Culotta and Hamer, 1989 CollagenasePinkert et al., 1987; Angel et al., 1987 Albumin Gene Pinkert et al.,1987, Tronche et al., 1989, 1990 α-Fetoprotein Godbout et al., 1988;Campere and Tilghman, 1989 t-Globin Bodine and Ley, 1987; Perez-Stableand Constantini, 1990 β-Globin Trudel and Constantini, 1987 e-fos Cohenet al., 1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985 InsulinEdlund et al., 1985 Neural Cell Adhesion Molecule Hirsch et al., 1990(NCAM) a_(1-Antitrypain) Latimer et al., 1990 H2B (TH2B) Histone Hwanget al., 1990 Mouse or Type I Collagen Ripe et al., 1989Glucose-Regulated Proteins Chang et al., 1989 (GRP94 and GRP78) RatGrowth Hormone Larsen et al., 1986 Human Serum Amyloid A (SAA) Edbrookeet al., 1989 Troponin I (TN I) Yutzey et al., 1989 Platelet-DerivedGrowth Factor Pech et al., 1989 Duchenne Muscular Dystrophy Klamut etal., 1990 SV40 Banerji et al., 1981; Moreau et al., 1981; Sleigh andLockett, 1985; Firak and Subramanian, 1986; Herr and Clarke, 1986; Imbraand Karin, 1986; Kadesch and Berg, 1986; Wang and Calame, 1986; Ondek etal., 1987; Kuhl et al., 1987 Schaffner et al., 1988 PolyomaSwartzendruber and Lehman, 1975; Vasseur et al., 1980; Katinka et al.,1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; deVilliers etal., 1984; Hen et al., 1986; Satake et al., 1988; Campbell andVillarreal, 1988 Retroviruses Kriegler and Botchan, 1982, 1983; Levinsonet al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze et al., 1986;Miksicek et al., 1986; Celander and Haseltine, 1987; Thiesen et al.,1988; Celander et al., 1988; Chol et al., 1988; Reisman and Rotter, 1989Papilloma Virus Campo et al., 1983; Lusky et al., 1983; Spandidos andWilkie, 1983; Spalholz et al., 1985; Lusky and Botchan, 1986; Cripe etal., 1987; Gloss et al., 1987; Hirochika et al., 1987, Stephens andHentschel, 1987; Glue et al., 1988 Hepatitis B Virus Bulla and Siddiqui,1986; Jameel and Siddiqui, 1986; Shaul and Ben-Levy, 1987; Spandau andLee, 1988 Human Immunodeficiency Virus Muesing et al., 1987; Hauber andCullan, 1988; Jakobovits et al., 1988; Feng and Holland, 1988; Takebe etal., 1988; Rowen et al., 1988; Berkhout et al., 1989; Laspia et al.,1989; Sharp and Marciniak, 1989; Braddock et al., 1989 CytomegalovirusWeber et al., 1984; Boshart et al., 1985; Foecking and Hofstetter, 1986Gibbon Ape Leukemia Virus Holbrook et al., 1987; Quinn et al., 1989

Another signal that may prove useful is a polyadenylation signal (hGH,BGH, SV40).

The use of internal ribosome binding sites (IRES) elements are used tocreate multigene, or polycistronic, messages. IRES elements are able tobypass the ribosome scanning model of 5′-methylated cap-dependenttranslation and begin translation at internal sites (Pelletier andSonenberg, 1988). IRES elements from two members of the picornavirusfamily (polio and encephalomyocarditis) have been described (Pelletierand Sonenberg, 1988), as well an IRES from a mammalian message (Macejakand Sarnow, 1991). IRES elements can be linked to heterologous openreading frames. Multiple open reading frames can be transcribedtogether, each separated by an IRES, creating polycistronic messages. Byvirtue of the IRES element, each open reading frame is accessible toribosomes for efficient translation. Multiple genes can be efficientlyexpressed using a single promoter/enhancer to transcribe a singlemessage.

As discussed above, in certain embodiments of the invention, a cell maybe identified and selected in vitro or in vivo by including a marker inthe expression construct. Such markers confer an identifiable change tothe cell permitting easy identification of cells containing theexpression construct. Usually, the inclusion of a drug selection markeraids in cloning and in the selection of transformants, for example,genes that confer resistance to neomycin, puromycin, hygromycin, DHFR,GPT, zeocin, tetracycline and histidinol are useful selectable markers.Alternatively, enzymes such as herpes simplex virus thymidine kinase(tk) or chloramphenicol acetyltransferase (CAT) may be employed.

The promoters and enhancers that control the transcription of proteinencoding genes in eukaryotic cells are composed of multiple geneticelements. The cellular machinery is able to gather and integrate theregulatory information conveyed by each element, allowing differentgenes to evolve distinct, often complex patterns of transcriptionalregulation.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV 40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between elements is flexible, so that promoterfunction is preserved when elements are inverted or moved relative toone another. In the tk promoter, the spacing between elements can beincreased to 50 bp apart before activity begins to decline. Depending onthe promoter, it appears that individual elements can function eitherco-operatively or independently to activate transcription.

Enhancers were originally detected as genetic elements that increasedtranscription from a promoter located at a distant position on the samemolecule of DNA. This ability to act over a large distance had littleprecedent in classic studies of prokaryotic transcriptional regulation.Subsequent work showed that regions of DNA with enhancer activity areorganized much like promoters. That is, they are composed of manyindividual elements, each of which binds to one or more transcriptionalproteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Aside from this operational distinction, enhancers and promoters arevery similar entities.

Promoters and enhancers have the same general function of activatingtranscription in the cell. They are often overlapping and contiguous,often seeming to have a very similar modular organization. Takentogether, these considerations suggest that enhancers and promoters arehomologous entities and that the transcriptional activator proteinsbound to these sequences may interact with the cellular transcriptionalmachinery in fundamentally the same way.

In any event, it will be understood that promoters are DNA elementswhich when positioned functionally upstream of a gene leads to theexpression of that gene. Most transgene constructs of the presentinvention are functionally positioned downstream of a promoter element.

D. ENGINEERING OF VIRAL VECTORS

In certain embodiments, the present invention further involves themanipulation of viral vectors. Such methods involve the use of a vectorconstruct containing, for example, a heterologous DNA encoding a gene ofinterest and a means for its expression, replicating the vector in anappropriate helper cell, obtaining viral particles produced therefrom,and infecting cells with the recombinant virus particles. The gene couldsimply encode a protein for which large quantities of the protein aredesired, i.e., large scale in vitro production methods. Alternatively,the gene could be a therapeutic gene, for example to treat cancer cells,to express immunomodulatory genes to fight viral infections, or toreplace a gene's function as a result of a genetic defect. In thecontext of the gene therapy vector, the gene will be a heterologous DNA,meant to include DNA derived from a source other than the viral genomewhich provides the backbone of the vector. Finally, the virus may act asa live viral vaccine and express an antigen of interest for theproduction of antibodies they are against. The gene may be derived froma prokaryotic or eukaryotic source such as a bacterium, a virus, ayeast, a parasite, a plant, or even an animal. The heterologous DNA alsomay be derived from more than one source, i.e., a multigene construct ora fusion protein. The heterologous DNA may also include a regulatorysequence which may be derived from one source and the gene from adifferent source.

1. Therapeutic Genes

p53 currently is recognized as a tumor suppressor gene (Montenarh,1992). High levels of mutant p53 have been found in many cellstransformed by chemical carcinogenesis, ultraviolet radiation, andseveral viruses, including SV40. The p53 gene is a frequent target ofmutational inactivation in a wide variety of human tumors and is alreadydocumented to be the most frequently-mutated gene in common humancancers (Mercer, 1992). It is mutated in over 50% of human NSCLC(Hollestein et al., 1991) and in a wide spectrum of other tumors.

The p53 gene encodes a 393-amino-acid phosphoprotein that can formcomplexes with host proteins such as large-T antigen and E1B. Theprotein is found in normal tissues and cells, but at concentrationswhich are generally minute by comparison with transformed cells or tumortissue. Interestingly, wild-type p53 appears to be important inregulating cell growth and division. Overexpression of wild-type p53 hasbeen shown in some cases to be anti-proliferative in human tumor celllines. Thus, p53 can act as a negative regulator of cell growth(Weinberg, 1991) and may directly suppress uncontrolled cell growth ordirectly or indirectly activate genes that suppress this growth. Thus,absence or inactivation of wild-type p53 may contribute totransformation. However, some studies indicate that the presence ofmutant p53 may be necessary for full expression of the transformingpotential of the gene.

Wild-type p53 is recognized as an important growth regulator in manycell types. Missense mutations are common for the p53 gene and are knownto occur in at least 30 distinct codons, often creating dominant allelesthat produce shifts in cell phenotype without a reduction tohomozygosity. Additionally, many of these dominant negative allelesappear to be tolerated in the organism and passed on in the germ line.Various mutant alleles appear to range from minimally dysfunctional tostrongly penetrant, dominant negative alleles (Weinberg, 1991).

Casey and colleagues have reported that transfection of DNA encodingwild-type p53 into two human breast cancer cell lines restores growthsuppression control in such cells (Casey et al., 1991). A similar effecthas also been demonstrated on transfection of wild-type, but not mutant,p53 into human lung cancer cell lines (Takahasi et al., 1992). p53appears dominant over the mutant gene and will select againstproliferation when transfected into cells with the mutant gene. Normalexpression of the transfected p53 is not detrimental to normal cellswith endogenous wild-type p53. Thus, such constructs might be taken upby normal cells without adverse effects. It is thus proposed that thetreatment of p53-associated cancers with wild-type p53 expressionconstructs will reduce the number of malignant cells or their growthrate. Furthermore, recent studies suggest that some p53 wild-type tumorsare also sensitive to the effects of exogenous p53 expression.

The major transitions of the eukaryotic cell cycle are triggered bycyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kinase 4(CDK4), regulates progression through the G₁ phase. The activity of thisenzyme may be to phosphorylate Rb at late G₁. The activity of CDK4 iscontrolled by an activating subunit, D-type cyclin, and by an inhibitorysubunit, e.g. p16^(INK4), which has been biochemically characterized asa protein that specifically binds to and inhibits CDK4, and thus mayregulate Rb phosphorylation (Serrano et al., 1993; Serrano et al.,1995). Since the p16^(INK4) protein is a CDK4 inhibitor (Serrano, 1993),deletion of this gene may increase the activity of CDK4, resulting inhyperphosphorylation of the Rb protein. p16 also is known to regulatethe function of CDK6.

p16^(INK4) belongs to a newly described class of CDK-inhibitory proteinsthat also includes p16^(B), p21^(WAF1, CIP1, SDI1), and p27^(KIP1). Thep16^(INK4) gene maps to 9p21, a chromosome region frequently deleted inmany tumor types. Homozygous deletions and mutations of the p16^(INK4)gene are frequent in human tumor cell lines. This evidence suggests thatthe p16^(INK)4 gene is a tumor suppressor gene. This interpretation hasbeen challenged, however, by the observation that the frequency of thep16^(INK4) gene alterations is much lower in primary uncultured tumorsthan in cultured cell lines (Caldas et al., 1994; Cheng et al., 1994;Hussussian et al., 1994; Kamb et al., 1994a; Kamb et al., 1994b; Mori etal., 1994; Okamoto et al., 1994; Nobori et al., 1995; Orlow et al.,1994; Arap et al., 1995). Restoration of wild-type p16^(INK4) functionby transfection with a plasmid expression vector reduced colonyformation by some human cancer cell lines (Okamoto, 1994; Arap, 1995).

C-CAM is expressed in virtually all epithelial cells (Odin and Obrink,1987). C-CAM, with an apparent molecular weight of 105 kD, wasoriginally isolated from the plasma membrane of the rat hepatocyte byits reaction with specific antibodies that neutralize cell aggregation(Obrink, 1991). Recent studies indicate that, structurally, C-CAMbelongs to the immunoglobulin (Ig) superfamily and its sequence ishighly homologous to carcinoembryonic antigen (CEA) (Lin and Guidotti,1989). Using a baculovirus expression system, Cheung et al. (1993a;1993b and 1993c) demonstrated that the first Ig domain of C-CAM iscritical for cell adhesion activity.

Cell adhesion molecules, or CAMs are known to be involved in a complexnetwork of molecular interactions that regulate organ development andcell differentiation (Edelman, 1985). Recent data indicate that aberrantexpression of CAMs may be involved in the tumorigenesis of severalneoplasms; for example, decreased expression of E-cadherin, which ispredominantly expressed in epithelial cells, is associated with theprogression of several kinds of neoplasms (Edelman and Crossin, 1991;Frixen et al., 1991; Bussemakers et al., 1992; Matsura et al., 1992;Umbas et al., 1992). Also, Giancotti and Ruoslahti (1990) demonstratedthat increasing expression of α₅β₁ integrin by gene transfer can reducetumorigenicity of Chinese hamster ovary cells in vivo. C-CAM now hasbeen shown to suppress tumor growth in vitro and in vivo.

Other tumor suppressors that may be employed according to the presentinvention include RB, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1,p73, BRCA1, VHL, FCC, MMAC1, MCC, p16, p21, p57, C-CAM, p27 and BRCA2.Inducers of apoptosis, such as Bax, Bak, Bcl-X_(s), Bik, Bid, Harakiri,Ad E1B, Bad and ICE-CED3 proteases, similarly could find use accordingto the present invention.

Various enzyme genes are of interest according to the present invention.Such enzymes include cytosine deaminase, hypoxanthine-guaninephosphoribosyltransferase, galactose-1-phosphate uridyltransferase,phenylalanine hydroxylase, glucocerbrosidase, sphingomyelinase,α-L-iduronidase, glucose-6-phosphate dehydrogenase, HSV thymidine kinaseand human thymidine kinase.

Hormones are another group of gene that may be used in the vectorsdescribed herein. Included are growth hormone, prolactin, placentallactogen, luteinizing hormone, follicle-stimulating hormone, chorionicgonadotropin, thyroid-stimulating hormone, leptin, adrenocorticotropin(ACTH), angiotensin I and II, β-endorphin, β-melanocyte stimulatinghormone (β-MSH), cholecystokinin, endothelin I, galanin, gastricinhibitory peptide (GIP), glucagon, insulin, lipotropins, neurophysins,somatostatin, calcitonin, calcitonin gene related peptide (CGRP),β-calcitonin gene related peptide, hypercalcemia of malignancy factor(1-40), parathyroid hormone-related protein (107-139) (PTH-rP),parathyroid hormone-related protein (107-111) (PTH-rP), glucagon-likepeptide (GLP-1), pancreastatin, pancreatic peptide, peptide YY, PHM,secretin, vasoactive intestinal peptide (VIP), oxytocin, vasopressin(AVP), vasotocin, enkephalinamide, metorphinamide, alpha melanocytestimulating hormone (alpha-MSH), atrial natriuretic factor (5-28) (ANF),amylin, amyloid P component (SAP-1), corticotropin releasing hormone(CRH), growth hormone releasing factor (GHRH), luteinizinghormone-releasing hormone (LHRH), neuropeptide Y, substance K(neurokinin A), substance P and thyrotropin releasing hormone (TRH).

Other classes of genes that are contemplated to be inserted into thevectors of the present invention include interleukins and cytokines.Interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11 IL-12, GM-CSF and G-CSF.

Examples of diseases for which the present viral vector would be usefulinclude, but are not limited to, adenosine deaminase deficiency, humanblood clotting factor IX deficiency in hemophilia B, and cysticfibrosis, which would involve the replacement of the cystic fibrosistransmembrane receptor gene. The vectors embodied in the presentinvention could also be used for treatment of hyperproliferativedisorders such as rheumatoid arthritis or restenosis by transfer ofgenes encoding angiogenesis inhibitors or cell cycle inhibitors.Transfer of prodrug activators such as the HSV-TK gene can be also beused in the treatment of hyperproliferative disorders, including cancer.

2. Antisense Constructs

Oncogenes such as ras, myc, neu, raf, erb, src, fms, jun, trk, ret, gsp,hst, bcl and abl also are suitable targets. However, for therapeuticbenefit, these oncogenes would be expressed as an antisense nucleicacid, so as to inhibit the expression of the oncogene. The term“antisense nucleic acid” is intended to refer to the oligonucleotidescomplementary to the base sequences of oncogene-encoding DNA and RNA.Antisense oligonucleotides, when introduced into a target cell,specifically bind to their target nucleic acid and interfere withtranscription, RNA processing, transport and/or translation. Targetingdouble-stranded (ds) DNA with oligonucleotide leads to triple-helixformation; targeting RNA will lead to double-helix formation.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. Antisense RNA constructs, or DNA encoding such antisense RNAs, maybe employed to inhibit gene transcription or translation or both withina host cell, either in vitro or in vivo, such as within a host animal,including a human subject. Nucleic acid sequences comprising“complementary nucleotides” are those which are capable of base-pairingaccording to the standard Watson-Crick complementarity rules. That is,that the larger purines will base pair with the smaller pyrimidines toform only combinations of guanine paired with cytosine (G:C) and adeninepaired with either thymine (A:T), in the case of DNA, or adenine pairedwith uracil (A:U) in the case of RNA.

As used herein, the terms “complementary” or “antisense sequences” meannucleic acid sequences that are substantially complementary over theirentire length and have very few base mismatches. For example, nucleicacid sequences of fifteen bases in length may be termed complementarywhen they have a complementary nucleotide at thirteen or fourteenpositions with only single or double mismatches. Naturally, nucleic acidsequences which are “completely complementary” will be nucleic acidsequences which are entirely complementary throughout their entirelength and have no base mismatches.

While all or part of the gene sequence may be employed in the context ofantisense construction, statistically, any sequence 17 bases long shouldoccur only once in the human genome and, therefore, suffice to specify aunique target sequence. Although shorter oligomers are easier to makeand increase in vivo accessibility, numerous other factors are involvedin determining the specificity of hybridization. Both binding affinityand sequence specificity of an oligonucleotide to its complementarytarget increases with increasing length. It is contemplated thatoligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ormore base pairs will be used. One can readily determine whether a givenantisense nucleic acid is effective at targeting of the correspondinghost cell gene simply by testing the constructs in vitro to determinewhether the endogenous gene's function is affected or whether theexpression of related genes having complementary sequences is affected.

In certain embodiments, one may wish to employ antisense constructswhich include other elements, for example, those which include C-5propyne pyrimidines. Oligonucleotides which contain C-5 propyneanalogues of uridine and cytidine have been shown to bind RNA with highaffinity and to be potent antisense inhibitors of gene expression(Wagner et al., 1993).

As an alternative to targeted antisense delivery, targeted ribozymes maybe used. The term “ribozyme” refers to an RNA-based enzyme capable oftargeting and cleaving particular base sequences in oncogene DNA andRNA. Ribozymes can either be targeted directly to cells, in the form ofRNA oligo-nucleotides incorporating ribozyme sequences, or introducedinto the cell as an expression construct encoding the desired ribozymalRNA. Ribozymes may be used and applied in much the same way as describedfor antisense nucleic acids.

3. Antigens for Vaccines

Other therapeutics genes might include genes encoding antigens such asviral antigens, bacterial antigens, fungal antigens or parasiticantigens. Viruses include picornavirus, coronavirus, togavirus,flavirvirus, rhabdovirus, paramyxovirus, orthomyxovirus, bunyavirus,arenvirus, reovirus, retrovirus, papovavirus, parvovirus, herpesvirus,poxvirus, hepadnavirus, and spongiform virus. Preferred viral targetsinclude influenza, herpes simplex virus 1 and 2, measles, small pox,polio or HIV. Pathogens include trypanosomes, tapeworms, roundworms,helminths. Also, tumor markers, such as fetal antigen or prostatespecific antigen, may be targeted in this manner. Preferred examplesinclude HIV env proteins and hepatitis B surface antigen. Administrationof a vector according to the present invention for vaccination purposeswould require that the vector-associated antigens be sufficientlynon-immunogenic to enable long term expression of the transgene, forwhich a strong immune response would be desired. Preferably, vaccinationof an individual would only be required infrequently, such as yearly orbiennially, and provide long term immunologic protection against theinfectious agent.

4. Control Regions

In order for the viral vector to effect expression of a transcriptencoding a therapeutic gene, the polynucleotide encoding the therapeuticgene will be under the transcriptional control of a promoter and apolyadenylation signal. A “promoter” refers to a DNA sequence recognizedby the synthetic machinery of the host cell, or introduced syntheticmachinery, that is required to initiate the specific transcription of agene. A polyadenylation signal refers to a DNA sequence recognized bythe synthetic machinery of the host cell, or introduced syntheticmachinery, that is required to direct the addition of a series ofnucleotides on the end of the mRNA transcript for proper processing andtrafficking of the transcript out of the nucleus into the cytoplasm fortranslation. The phrase “under transcriptional control” means that thepromoter is in the correct location in relation to the polynucleotide tocontrol RNA polymerase initiation and expression of the polynucleotide.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work; have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription.

E. PHARMACEUTICAL COMPOSITIONS

In certain embodiments, the present invention also concerns formulationsof a viral composition for administration to a mammal. It will also beunderstood that, if desired, the viral compositions disclosed herein maybe administered in combination with other agents as well, such as, e.g.,various pharmaceutically-active agents. As long as the compositions donot cause a significant adverse effect upon contact with the targetcells or host tissues, there is virtually no limit to other componentswhich may also be included.

The formulation of pharmaceutically-acceptable excipients and carriersolutions are well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation.

1. Injectable Compositions and Delivery

The pharmaceutical compositions disclosed herein may be administeredparenterally, intravenously, intramuscularly, or even intraperitoneallyas described in U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 andU.S. Pat. No. 5,399,363 (each specifically incorporated herein byreference in its entirety). Solutions of the active compounds as freebase or pharmacologically acceptable salts may be prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions may also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

Typically, these formulations may contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared in such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial adantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug release capsules and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

2. Oral Compositions and Delivery

Alternatively, the pharmaceutical compositions disclosed herein may bedelivered via oral administration to an animal, and as such, thesecompositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

The active compounds may even be incorporated with excipients and usedin the form of ingestible tablets, buccal tables, troches, capsules,elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al.,1997; Hwang et al., 1998; U.S. Pat. No. 5,641,515; U.S. Pat. No.5,580,579 and U.S. Pat. No. 5,792,451, each specifically incorporatedherein by reference in its entirety). The tablets, troches, pills,capsules and the like may also contain the following: a binder, as gumtragacanth, acacia, cornstarch, or gelatin; excipients, such asdicalcium phosphate; a disintegrating agent, such as corn starch, potatostarch, alginic acid and the like; a lubricant, such as magnesiumstearate; and a sweetening agent, such as sucrose, lactose or saccharinmay be added or a flavoring agent, such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup of elixir may contain the active compounds sucrose as a sweeteningagent methyl and propylparabens as preservatives, a dye and flavoring,such as cherry or orange flavor. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompounds may be incorporated into sustained-release preparation andformulations.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as those containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, including: gels,pastes, powders and slurries, or added in a therapeutically effectiveamount to a paste dentifrice that may include water, binders, abrasives,flavoring agents, foaming agents, and humectants, or alternativelyfashioned into a tablet or solution form that may be placed under thetongue or otherwise dissolved in the mouth.

3. Nasal Delivery

The administration of agonist pharmaceutical compositions by intranasalsprays, inhalation, and/or other aerosol delivery vehicles is alsoconsidered. Methods for delivering genes, nucleic acids, and peptidecompositions directly to the lungs via nasal aerosol sprays has beendescribed e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212(each specifically incorporated herein by reference in its entirety),and delivery of drugs using intranasal microparticle resins (Takenaga etal., 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No.5,725,871, specifically incorporated herein by reference in itsentirety) are also well-known in the pharmaceutical arts. Likewise,transmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045 (specificallyincorporated herein by reference in its entirety).

F. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Materials and Methods

Lyophilizer

A Dura-stop μp lyophilizer (FTSsystems) with in process sampleretrieving device was used. The lyophilizer is equipped with boththermocouple vacuum gauge and capacitance manometer for vacuummeasurement. Condenser temperature is programmed to reach to −80° C.Vials were stoppered at the end of each run with a build-in mechanicalstoppering device.

Residual Moisture Measurement

Residual moisture in freeze dried product was analyzed by a Karl-Fishertype coulometer (Mettler DL37, KF coulometer).

HPLC Analysis

HPLC analysis of samples was done on a Beckman Gold HPLC system.

Vials and Stoppers

Borosilicate 3 ml with 13 mm opening lyo vials and their correspondingbutyl rubber stoppers (both from Wheaton) were used for bothlyophilization and liquid formulation development. The stoppered vialswere capped with Flip-off aluminum caps using a capping device (LW312Westcapper, The West Company).

Example 2 Lyophilization Initial Cycle and Formulation Development

There are three main process variables that can be programmed to achieveoptimal freeze-drying. Those are shelf temperature, chamber pressure,and lyophilization step duration time. To avoid cake collapse, shelftemperature need to be set at temperatures 2-3° C. below the glasstransition or eutectic temperature of the frozen formulation. Both theglass transition and eutectic temperatures of a formulation can bedetermined by differential scanning calorimetry (DSC) analysis. Chamberpressure is generally set at below the ice vapor pressure of the frozenformulation. The ice vapor pressure is dependent on the shelftemperature and chamber pressure. Too high a chamber pressure willreduce the drying rate by reducing the pressure differential between theice and the surrounding, while too low a pressure will also slow downdrying rate by reducing the heat transfer rate from the shelf to thevials. The development of a lyophilization cycle is closely related withthe formulation and the vials chosen for lyophilization. The goal atthis stage was to develop a somewhat conservative cycle to be able tosuccessfully freeze dry a number of different formulations. Thedeveloped cycles and formulations will be further optimized when virusesare formulated in the formulations. Formulation excipient selection wasbased on the classical excipients found in most lyophilizedpharmaceuticals. The excipients in a lyophilization formulation shouldprovide the functions of bulking, cryoprotection, and lyoprotection. Theexcipients chosen were mannitol (bulking agent), sucrose (cryo- andlyoprotectant), and human serum albumin (HSA, lyoprotectant). Theseexcipients were formulated in 10 mM Tris+1 mM MgCl₂, pH=7.50 at variouspercentages and filled into the 3 ml vials at a fill volume of 1 ml. Tostart with, a preliminary cycle was programmed to screen a variety offormulations based on the criteria of residual moisture and physicalappearance after drying. The cycle used is plotted in FIG. 1. Extensivescreening was carried out by variation of the percentages of theindividual excipients. Table 4 shows briefly some of the results.

TABLE 4 Evaluation of Different Formulations Under the Same CycleFormulation M %/S %/HSA % Appearance Moisture (% weight) 10/5/0.5  goodcake 0.89 5/5/0.5 good cake 1.5 3/5/0.5 loose cake (partial collapse)3.4 1/5/0.5 no cake (collapse) 6.4

The results suggest that a minimum amount of 3% mannitol is required inthe formulation in order to achieve pharmaceutically elegant cake. Thepercentages of sucrose in the formulation were also examined. Nosignificant effect on freeze-drying was observed at sucroseconcentrations of ≦10%. HSA concentration was kept constant to 0.5%during the initial screening stage.

After the evaluation of the formulations, freeze-drying cycle wasoptimized by changing the shelf temperature, chamber vacuum and theduration of each cycle step. Based on the extensive cycle optimization,the following cycle (cycle #14) was used for further viruslyophilization development.

-   1. Load sample at room temperature onto shelf.-   2. Set shelf temperature to −45° C. and freeze sample. Step time 2    h.-   3. Set shelf temperature at −45° C., turn vacuum pump and set vacuum    at 400 mT. Step time 5 h.-   4. Set shelf temperature at −35° C., set vacuum at 200 mT. Step time    13 h.-   5. Set shelf temperature at −22° C., set vacuum at 100 mT. Step time    15 h.-   6. Set shelf temperature at −10° C., set vacuum at 100 mT. Step time    5 h.-   7. Set shelf temperature at 10° C., set vacuum at 100 mT. Step time    4 h.-   8. Vial stoppering under vacuum.

Example 3 Cycle and Formulation Development with Virus in Formulation

Effect of Sucrose Concentration in Formulation. Cycle and formulationwere further optimized according to virus recovery after lyophilizationanalyzed by both HPLC and plaque forming unit (PFU) assays. Table 5shows the virus recoveries immediate after drying in differentformulations using the above drying cycle. Variation of the percentageof sucrose in the formulation had significant effect on virusrecoveries.

TABLE 5 Recoveries of Virus After Lyophilization Formulation M %/S %/HSA% Appearance Residual moisture Recovery (%) 6/0/0.5 good cake 0.44%  06/3.5/0.5 good cake 2.2% 56 6/5/0.5 good cake 2.5% 81 6/6/0.5 good cake2.7% 120 6/7/0.5 good cake 2.8% 120 6/8/0.5 good cake 3.3% 93 6/9/0.5good cake 3.7% 120

Residual moisture in the freeze-dried product increased as the sucrosepercentage increased. A minimum sucrose concentration of 5% is requiredin the formulation to maintain a good virus recovery afterlyophilization. Similar sucrose effects in formulation that had 5%instead of 6% mannitol were observed. However, good virus recoveryimmediately after drying does not necessary support a good long-termstorage stability. As a result, formulations having 4 different sucroseconcentrations of 6, 7, 8, and 9%, were incorporated for furtherevaluation.

Effect of HSA in Formulation. The contribution of HSA concentrations inthe formulation on virus recovery after drying was examined using thesame freeze drying cycle. Table 6 shows the results.

TABLE 6 Effects of HSA Concentration on Lyophilization Formulation M %/S%/HSA % Appearance Residual moisture Recovery (%) 6/7/0 Good cake 0.9883 6/7/0.5 Good cake 1.24 120 6/7/2 Good cake 1.5 110 6/7/5 Good cake1.7 102

The results indicate that inclusion of HSA in the formulation hadpositive effect on virus recovery after drying. Concentrations higherthan 0.5% did not further improve the virus recovery post drying. As aresult, 0.5% HSA is formulated in all the lyophilization formulations.

Cycle Optimization. As indicated in Table 5, relatively high residualmoistures were present in the dried product. Although there has not beena known optimal residual moisture for freeze dried viruses, it could bebeneficial for long term storage stability to further reduce theresidual moisture in the dried product. After reviewing of the dryingcycle, it was decided to increase the secondary drying temperature from10° C. to 30° C. without increasing the total cycle time. As indicatedin Table 7, significant reduction in residual moisture had been achievedin all the formulations without negative effects on virus recoveries.With the improved drying cycle, residual moisture was less than 2% inall the formulations immediately after drying. It is expected that thereduced residual moisture will improve the long-term storage stabilityof the dried product.

TABLE 7 Effects of Secondary Drying Temperature on LyophilizationSecondary drying Secondary drying at 10° C. at 30° C. FormulationResidual moisture Recovery Residual M %/S %/HSA % (w %) (%) moistureRecovery 6/6/0.5 2.2 100 0.8 93 6/7/0.5 2.5 86 1.1 100 6/8/0.5 2.7 831.3 87 6/9/0.5 3.3 93 1.5 86 5/6/0.5 2.3 110 1.0 94 5/7/0.5 2.7 88 1.285 5/8/0.5 3.5 97 1.6 88 5/9/0.5 4 90 1.9 86

N₂ Backfilling (Blanketing). Lyophilization was done similarly as aboveexcept that dry N₂ was used for gas bleeding for pressure control duringthe drying and backfilling at the end of the cycle. At the end of adrying run, the chamber was filled with dry N₂ to about 80% atmosphericpressure. Subsequently, the vials were stoppered. No difference wasnoticed between the air and N₂ blanketing runs immediate after drying.However, if oxygen present in the vial during air backfilling causesdamaging effect (oxidation) on the virus or excipients used duringlong-term storage, backfilling with dry N₂ is likely to ameliorate thedamaging effects and improve long term storage stability of the virus.

Removal of Glycerol From Formulation. During the preparation of viruscontaining formulations, stock virus solution was added to thepre-formulated formulations at a dilution factor of 10. Because of thepresence of 10% glycerol in the stock virus solution, 1% glycerol wasintroduced into the formulations. To examine any possible effect of thepresence of 1% glycerol on lyophilization, a freeze drying run wasconducted using virus diafiltered into the formulation of 5% (M)/7%(S)/0.5% (HSA). Diafiltration was done with 5 vol. of buffer exchangeusing a constant volume buffer exchange mode to ensure adequate removalof residual glycerol (99% removal). After diafiltration, virus solutionwas filled into vials and then lyophilized similarly. Table 8 shows thelyophilization results.

TABLE 8 Lyophilization without Glycerol Formulation M %/S %/HSA %Residual moisture Recovery (%) 5/7/0.5 1.0 80

No significant difference after freeze drying was observed betweenformulations with and without 1% glycerol. Possible implications of thischange on long term storage will be evaluated.

Example 4 Long Term Storage Stability Study

Adp53 virus lyophilized under different formulations and differentcycles was placed at −20° C., 4° C., and room temperature (RT) underdark for long term storage stability evaluation. Parameters measuredduring the stability study were PFU, HPLC viral particles, residualmoisture, and vacuum inside vial. The plan is to be able to evaluatevirus stability at various conditions for up to one-year storage. Table6 shows the data after 12-month storage with secondary drying at 10° C.without N₂ blanketing. Lyophilized virus is stable at both −20° C. and4° C. storage for up to 12 months. However, virus was not stable at roomtemperature storage. More than 50% loss in infectivity was observed atRT after 1-month storage. The reason for the quick loss of infectivityat RT is not clear. However, it is likely that RT is above the glasstransition temperature of the dried formulation and resulting in theaccelerated virus degradation. A differential scanning caloremitry (DSC)analysis of the formulation could provide very useful information.Pressure change inside the vials during storage was not detected, whichindicates that the vials maintained their integrity. The slight increasein residual moisture during storage can be attributed to the release ofmoisture from the rubber stopper into the dried product.

TABLE 9 Secondary Drying at 10° C. Formulation Set 10 (6-9) PFU × 10⁹/mlHPLC Viral Particle (×10¹⁰/ml) Water Content (W %) Date* Set 10-6 Set10-7 Set 10-8 Set 10-9 Set 10-6 Set 10-7 Set 10-8 Set 10-9 Set 10-6 Set10-7 Set 10-8 Set 10-9 Apr. 11, 1997 5.5 6.0 5.8 6.5 24.5 24.6 24.9 26.72.2 2.5 2.7 3.3 May 15, 1997^(a) 7.6 7.1 7.5 8.1 22.4 25.6 26.8 28.5 2.22.5 2.8 3.3 May 15, 1997^(b) 6.6 6.3 6.5 10.0 22.0 23.0 24.0 27.5 2.42.6 3.0 3.4 May 15, 1996^(c) 7.1 7.1 6.7 3.3 14.5 16.5 6.2 4.2 2.7 2.93.2 3.5 Jul. 18, 1997^(a) 6.8 6.4 6.8 7.2 28.7 28.9 28.6 31.2 2.3 2.52.8 3.3 Jul. 18, 1997^(b) 6.0 5.8 7.3 9.0 25.0 26.6 27.6 31.1 2.5 2.83.0 3.6 Jul. 18, 1997^(c) 1.2 0.8 4.0 1.4 0.9 1.8 0.7 0.7 2.7 2.9 3.03.4 Oct. 22, 1997^(a) 7.9 7.5 7.9 7.8 25.5 25.0 25.4 26.2 2.4 2.6 2.83.1 Oct. 22, 1997^(b) 6.8 6.8 5.8 8.0 22.0 23.0 24.7 24.2 2.7 2.9 3.23.6 Oct. 22, 1997^(c) <0.01 <0.01 <0.01 <0.01 N.D. N.D. N.D. N.D. 2.72.9 3.1 3.4 Apr. 16, 1998^(a) 6.0 5.8 7.1 7.2 19.3 20.3 23.5 26.1 2.42.6 3.0 3.4 Apr. 16, 1998^(b) 5.4 7.2 6.1 6.3 21.7 22.8 22.9 24.6 2.93.1 3.3 3.8 Apr. 16, 1998^(c) 0.0003 0.001 0.0007 0.001 N.D. N.D. N.D.N.D. 2.7 2.9 3.1 3.4 *Temp. ^(a)(−20° C.) ^(b)(4° C.) ^(c)(r.t.)Controls PFU × 10⁹/ml HPLC Viral Particle (×10¹⁰/ml) Date Set 10-6 Set10-7 Set 10-8 Set 10-9 Set 10-6 Set 10-7 Set 10-8 Set 10-9 Apr. 11, 19975.5 7.0 7.0 7.0 35.5 35.8 36.0 36.9 N.D.: not detectable Formulation set10: 6%-mannitol. 0.5% HSA, 1% glycerol and different percentages ofsucrose in 10 mM-tris buffer pH = 7.5, 1 mM MgCl₂ Formulation Set 11(6-9) PFU × 10⁹/ml HPLC Viral Particle (×10¹⁰/ml) Water Content (W %)Date* Set 11-6 Set 11-7 Set 11-8 Set 11-9 Set 11-6 Set 11-7 Set 11-8 Set11-9 Set 11-6 Set 11-7 Set 11-8 Set 11-9 May 2, 1997 7.0 6.0 6.3 5.828.5 28.8 28.4 29.5 2.3 2.7 3.5 4.0 Jun. 20, 1997^(a) 6.2 6.6 6.9 6526.4 25.0 27.0 27.3 2.2 2.8 34 4.6 Jun. 20, 1997^(b) 6.1 6.0 6.5 6.524.1 22.1 25.6 26.6 2.5 2.8 3.5 4.8 Jun. 20, 1997^(c) 3.3 3.0 1.0 <0.120.5 17.4 5.2 9.1 2.7 3.1 3.5 4.7 Aug. 18, 1997^(a) 8.0 7.2 7.5 7.6 21.621.8 25.3 24.9 2.3 2.8 3.7 4.9 Aug. 18, 1997^(b) 8.0 7.3 8.0 8.0 22.722.7 24.9 25.0 2.6 3 3.9 4.2 Aug. 18, 1997^(c) <0.1 <0.1 <0.1 <0.1 N.D.N.D. 0.2 13.1 2.7 3.0 3.5 4.4 Oct. 22, 1997^(a) 79 7.5 7.9 6.7 21.0 22.025.1 24.0 2.4 3.0 3.9 4.4 Oct. 22, 1997^(b) 6.0 6.9 6.8 7.3 21.4 22.023.1 23.1 2.6 3.0 3.3 4.6 Oct. 22, 1997^(c) <0.01 <0.01 <0.01 <0.015N.D. N.D. N.D. 9.0 2.7 2.9 3.9 5.0 May 8, 1998^(a) 8.3 7.5 8.0 8.7 19.018.2 19.9 21.1 2.6 3.1 4.0 4.6 May 8, 1998^(b) 7.0 7.1 7.8 6.5 17.3 17.118.2 17.8 2.8 3.2 4.1 5.1 May 8, 1998^(c) 0.00033 0.000065 0.000450.000016 N.D. N.D. N.D. N.D. 2.7 2.9 4.0 4.9 *Temp. ^(a)(−20° C.)^(b)(4° C.) ^(c)(R.T.) Controls PFU × 10⁹/ml HPLC Viral Particle(×10¹⁰/ml) Date Set 11-6 Set 11-7 Set 11-8 Set 11-9 Set 11-6 Set 11-7Set 11-8 Set 11-9 May 2, 1997 6.4 6.8 6.5 6.5 37.7 35.7 37.3 36.0 N.D.:not detectable Formulation set 11: 5%-mannitol, 0.5% HSA, 1%-glyceroland different percentages of sucrose in 10 mM-tris buffer (pH = 7.5, 1mM MgCl₂) F11-(6-9)R1-S

TABLE 10 Secondary Drying at 30° C. Without N₂ Blanketing FormulationSet 10 (6-9) PFU × 10⁹/ml HPLC Viral Particle (×10¹⁰/ml) Water Content(W %) Date* Set 10-6 Set 10-7 Set 10-8 Set 10-9 Set 10-6 Set 10-7 Set10-8 Set 10-9 Set 10-6 Set 10-7 Set 10-8 Set 10-9 May 15, 1997 6.5 5.66.1 6.0 18.0 18.6 21.9 23.3 0.8 1.1 1.3 1.5 Jun. 20, 1997^(b) 5.4 5.65.5 5.5 14.6 14.9 17.2 16.6 0.8 1.2 1.5 1.6 Jun. 20, 1997^(c) 4.5 5.05.5 6.0 10.8 11.8 15.0 15.4 1.3 1.4 1.6 1.9 Aug. 18, 1997^(b) 7.0 6.76.8 7.0 15.3 17.1 17.9 17.7 1.3 1.5 1.5 1.7 Aug. 18, 1997^(c) 2.4 2.24.8 5.8 4.3 7.2 11.7 14.2 1.3 1.6 1.7 2.1 Nov. 20, 1997^(b) 5.5 5.5 5.35.7 21.9 21.9 27.2 26.4 1.1 1.4 1.6 1.9 Nov. 20, 1997^(c) 0.5 0.9 2.33.1 1.5 6.3 8.8 13.5 1.3 1.7 1.8 2.2 May 14, 1998^(ab) 4.9 4.7 5.4 6.59.7 11.9 12.6 14.2 1.2 1.6 2.2 1.4 May 14, 1998^(c) 0.000006 0.000060.00004 0.000024 N.D. N.D. N.D. N.D. 1.4 1.6 1.3 2.0 *Temp. ^(ab)(4° C.)^(c)(R.T.) Controls PFU × 10⁹/ml HPLC Viral Particle (×10¹⁰/ml) Date Set10-6 Set 10-7 Set 10-8 Set 10-9 Set 10-6 Set 10-7 Set 10-8 Set 10-9 May15, 1997 7.0 5.6 7.0 7.0 31.2 30.6 31.6 31.4 Formulation set 10:6%-mannitol, 0.5% HSA, 1%-glycerol and different percentages of sucrosein 10 mM-tris buffer (pH = 7.5, 1 mM MgCl₂) F10(6-9)R2-S Formulation Set11 (6-9) PFU × 10⁹/ml HPLC Viral Particle (×10¹⁰/ml) Water Content (W %)Date* Set 11-6 Set 11-7 Set 11-8 Set 11-9 Set 11-6 Set 11-7 Set 11-8 Set11-9 Set 11-6 Set 11-7 Set 11-8 Set 11-9 May 22, 1997 7.5 6.3 7.3 6.517.4 16.6 20.3 24.7 1.0 1.2 1.6 1.9 Jun. 20, 1997^(b) 5.5 6.3 6.0 7.514.8 16.1 17.5 21.1 1.2 1.3 1.7 1.8 Jun. 20, 1997^(c) 5.0 6.0 6.0 7.512.6 14.9 17.2 20.3 1.4 1.6 1.9 2.0 Aug. 18, 1997^(b) 6.3 6.7 68 7.515.7 17.2 18.5 22.6 1.2 1.5 1.8 1.9 Aug. 18, 1997^(c) 3.3 4.5 5.5 7.07.4 10.5 15.8 21.2 1.6 1.7 1.9 2.2 Nov. 20, 1997^(b) 5.3 5.6 5.3 6.622.6 26.4 30.0 35.0 1.2 1.4 1.9 1.9 Nov. 20, 1997^(c) 0.8 1.9 3.0 0.13.2 9.6 18.3 1.3 1.6 1.7 2.0 2.1 May 14, 1998^(b) 6.7 7.2 6.9 7.6 12.413.9 15.5 18.5 1.3 1.6 2.0 2.2 May 14, 1998^(c) 0.0013 0.00005 0.000310.00045 N.D. N.D. N.D. N.D. 1.6 1.8 1.6 2.0 *Temp. ^(a)(−20° C.) ^(b)(4°C.) ^(c)(R.T.) Controls PFU × 10⁹/ml HPLC Viral Particle (×10¹⁰/ml) DateSet 11-6 Set 11-7 Set 11-8 Set 11-9 Set 11-6 Set 11-7 Set 11-8 Set 11-9May 22, 1997 8.0 7.4 8.3 7.6 26.7 27.6 27.5 32.4 Formulation set 11:5%-mannitol, 0.5% HSA, 1%-glycerol and different percentages of sucrosein 10 mM-tris buffer (pH = 7.5, 1 mM MgCl₂) F11-(6-9)R2-S

TABLE 11 Secondary Drying at 30° C. With N₂ Blanketing Formulation Set10 (6-9) + Adp53 PFU × 10⁹/ml HPLC Viral Particle (×10¹⁰/ml) WaterContent (W %) Date* Set 10-6 Set 10-7 Set 10-8 Set 10-9 Set 10-6 Set10-7 Set 10-8 Set 10-9 Set 10-6 Set 10-7 Set 10-8 Set 10-9 Jun. 13, 19973.4 4.3 4.1 4.2 16.0 16.5 16.1 18.1 0.8 1.1 1.3 1.4 Jul. 18, 1997^(b)6.3 6.3 6.0 6.0 17.9 19.5 19.9 20.6 0.9 1.2 1.4 1.6 Jul. 18, 1997^(c)4.1 5.5 5.0 5.5 11.4 15.5 18.2 20.6 1.2 1.4 1.7 1.8 Sep. 16, 1997^(b)4.2 5.5 4.5 5.1 15.3 16.1 16.4 17.8 1.0 1.3 1.5 1.7 Sep. 16, 1997^(c)0.7 1.2 5.0 4.0 2.9 5.0 9.5 13.0 1.3 1.5 1.8 2.0 Dec. 4, 1997^(b) 5.55.3 5.4 5.9 16.1 16.2 18.1 18.5 1.1 1.4 1.6 1.7 Dec. 4, 1997^(c) 0.3 0.52.5 3.4 N.D. 1.7 4.7 8.8 1.4 1.6 1.8 2.0 Jun. 29, 1998^(ab) 3.8 5.1 5.35.4 10.6 10.8 12.0 12.9 1.3 1.5 1.8 1.9 Jun. 29, 1998^(c) 0.000030.00006 0.0001 0.0001 N.D. N.D. N.D. N.D. 1.4 1.6 1.7 1.8 *Temp.^(ab)(4° C.) ^(c)(R.T.) Controls PFU × 10⁹/ml HPLC Viral Particle(×10¹⁰/ml) Date Set 10-6 Set 10-7 Set 10-8 Set 10-9 Set 10-6 Set 10-7Set 10-8 Set 10-9 Jun. 13, 1997 4.7 3.8 5.5 6.2 26.0 26.2 27.4 27.5Formulation set 10: 6%-mannitol, 0.5% HSA, 1%-glycerol and differentpercentages of sucrose in 10 mM-tris buffer (pH: 7.5, 1 mM MgCl₂)F11-(6-9)R3-S Continued Formulation set 11 (6-9) + Adp53 PFU × 10⁹/mlHPLC Viral Particle (×10¹⁰/ml) Water Content (W %) Date* Set 11-6 Set11-7 Set 11-8 Set 11-9 Set 11-6 Set 11-7 Set 11-8 Set 11-9 Set 11-6 Set11-7 Set 11-8 Set 11-9 Jun. 13, 1997 3.4 4.2 3.6 4.4 16.1 16.3 18.4 19.30.9 1.3 1.8 1.9 Jul. 18, 1997^(b) 5.5 6.2 6.5 6.2 18.0 19.5 23.0 23.91.0 1.4 1.8 2.1 Jul. 18, 1997^(c) 3.7 6.0 6.7 7.3 13.7 18.7 21.8 22.81.3 1.7 2.0 2.2 Sep. 16, 1997^(b) 3.9 4 4.6 6 15.6 17.3 19.5 20.6 1.31.5 1.9 2.1 Sep. 16, 1997^(c) 0.78 2.2 4.0 5.3 3.6 6.8 13.8 14.6 1.5 1.92.3 2.4 Dec. 4, 1997^(b) 4.6 5.3 8.0 6.1 15.7 18.2 21.4 21.6 1.2 1.6 2.12.2 Dec. 4, 1997^(c) 0.4 0.6 0.3 0.01 N.D. N.D. 1.7 N.D. 1.6 1.8 2.1 2.1Jun. 29, 1998^(ab) 4.9 5.0 5.4 6.4 11.4 14.2 13.7 16.0 1.5 1.7 2.1 2.6Jun. 29, 1998^(c) 0.0001 0.00015 0.00085 0.0012 N.D. N.D. N.D. N.D. 1.61.7 2.2 2.3 *Temp. ^(ab)(4° C.) ^(c)(R.T.) Controls PFU × 10⁹/ml HPLCViral Particle (×10¹⁰/ml) Date Set 11-6 Set 11-7 Set 11-8 Set 11-9 Set11-6 Set 11-7 Set 11-8 Set 11-9 Jun. 13, 1997 4.5 5.0 4.0 5.0 26.5 26.926.6 27.1 Formulation set 11: 6%-mannitol, 0.5% HSA, 1%-glycerol anddifferent percentages of sucrose in 10 mM-tris buffer (pH = 7.5, 1 mMMgCl₂) F11-(6-9)R3-S

TABLE 12 Aqueous Formulation Set #1 Date (Storage Conds.) 10%-G 5%-S +5%-HSA 5%-S + 1%-PEG 5%-T + 1%-PEG PFU × 10⁹/ml Aug. 1, 1997 5.8 4.7 4.34.4 Aug. 28, 1997 (4° C., N₂) 5.8 5.8 6.4 6.3 Aug. 28, 1997 (4° C.,.Air) 5.0 5.9 6.0 5.9 Aug. 28, 1997 (R.T., N₂) 4.4 4.8 5.0 6.0 Aug. 28,1997 (R.T., Air) 4.3 5.0 5.0 5.6 Oct. 30, 1997 (4° C., N₂) 3.8 4.0 4.73.8 Oct. 30, 1997 (4° C. Air) 3.0 4.1 3.7 4.7 Oct. 30, 1997 (R.T., N2)1.5 3.4 3.5 3.6 Oct. 30, 1997 (R.T., Air) 1.5 3.6 2.2 3.1 Jan. 12, 1998(4° C., N₂) 3.2 4.1 3.3 3.4 Jan. 12, 1998 (4° C., Air) 1.5 3.8 3.9 3.4Jan. 12, 1998 (R.T., N₂) 0.1 1.4 0.7 0.7 Jan. 12, 1998 (R.T., Air) 0.41.6 1.0 0.4 Apr. 30, 1998 (4° C., N₂) 0.08 4.3 4.0 5.3 Apr. 30, 1998 (4°C., Air) 1.5 3.6 4.4 4.5 Apr. 30, 1998 (R.T., N₂) 0.0025 0.23 0.11 0.174/30.98 (R.T., Air) 0.0015 0.21 0.063 0.007 Feb. 5, 1999 (4° C., N₂)0.0005 5.8 4.1 3.9 Feb. 5, 1999 (4° C., Air) 0.02 4.7 4.3 4.5 Feb. 5,1999 (R.T., N₂) <10² 0.0007 <10⁴ 0.0002 Feb. 5, 1999 (R.T., Air) 2 × 10²0.0002 0.0003 2 × 10³ HPLC Viral Particle (×10¹⁰/ml) Aug. 1, 1997 16.914.5 16.1 16.7 Aug. 28, 1997 (4° C., N₂) 13.3 14.9 13.8 13.4 Aug. 28,1997 (4° C., Air) 12.9 14.2 12.9 12.9 Aug. 28, 1997 (R.T., N₂) 12.6 14.513.5 12.9 Aug. 28, 1997 (R.T., Air) 12.3 13.7 13.0 13.0 Oct. 30, 1997(4° C., N₂) 14.0 15.5 14.7 14.8 Oct. 30, 1997 (4° C., Air) 12.6 14.914.3 14.4 Oct. 30, 1997 (R.T., N₂) 13.8 15.1 14.6 14.4 Oct. 30, 1997(R.T., Air) 12.7 14.7 14.8 14.4 Jan. 12, 1998 (4° C., N₂) 7.3 11.1 9.59.5 Jan. 12, 1998 (4° C., Air) 7.7 10.8 10.2 10.0 Jan. 12, 1998 (R.T.,N2) 10.0 10.8 11.1 10.4 Jan. 12, 1998 (R.T., Air) 9.9 11.0 10.0 10.4Apr. 30, 1998 (4° C., N₂) 5.1 12.3 12.3 12.1 Apr. 30, 1998 (4° C., Air)5.0 11.6 11.8 11.9 Apr. 30, 1998 (R.T., N₂) 11.1 12.3 12.6 12.5 4/30.98(R.T., Air) 11.0 12.4 12.3 11.0 Feb. 5, 1999 (4° C., N₂) 3.4 5.8 11.411.0 Feb. 5, 1999 (4° C., Air) 3.9 7.1 11.0 11.2 Feb. 5, 1999 (R.T., N₂)10.1 7.9 8.5 10.9 Feb. 5, 1999 (R.T., Air) 9.7 7.1 10.3 9.3 G: glycerolS: sucrose PEG: PEG-3500 T: trehalose Glycerol: 10% glycerol in DPBSbuffer Other formulations are in 10 mM-tris buffer with 0.15 M-NaCl and1 mM-MgCl₂ (pH = 8.2).

TABLE 13 Aqueous Formulation Set #2 PFU × 10⁹/ml Date (Temp.) AQF2-1AQF2-2 AQF2-3 AQF2-4 AQF2-5 AQF2-6 AQF2-7* Sep. 25, 1997 2.8 2.8 2.8 3.02.8 2.8 2.7 Nov. 05, 1997 (4° C.) 2.3 3.2 2.4 3.6 2.7 2.0 3.6 Nov. 05,1997 (R.T.) 1.4 1.9 1.3 1.5 2.4 2.5 3.1 Dec. 12, 1997 (4° C.) 2.2 0.12.4 2.7 2.1 2.1 3.2 Jan. 09, 1998 (R.T.) 1.2 0.1 0.2 1.2 0.2 0.1 1.3 PFU× 10⁹/ml Date (Temp.) AQF2-8* AQF2-9* AQF2-10* AQF2-11* AQF2-12 Sep. 25,1997 2.8 2.7 3.3 3.1 2.7 Nov. 05, 1997 (4° C.) 3.8 2.7 3.0 3.5 2.5 Nov.05, 1997 (R.T.) 3.3 3.1 4.1 2.8 1.1 Dec. 12, 1997 (4° C.) 2.1 3.0 3.03.4 2.9 Jan. 09, 1998 (R.T.) 1.1 0.2 0.1 20 1.1 *Gave better recovery.HPLC viral particle (×10¹⁰/ml) Date (Temp.) AQF2-1 AQF2-2 AQF2-3 AQF2-4AQF2-5 AQF2-6 AQF2-7 Sep. 25, 1997 10.9 9.6 9.7 11.3 10.7 10.6 10.9 Nov.05, 1997 (4° C.) 7.9 7.6 8.7 8.8 8.9 7.5 8.6 Nov. 05, 1997 (R.T.) 8.26.6 7.6 8.6 7.7 9.3 9.0 Dec. 12, 1997 (4° C.) 6.7 1.5 8.0 6.9 5.2 7.57.5 Dec. 17, 1997 (R.T.) 7.0 1.2 7.0 7.5 4.1 7.1 7.0 HPLC viral particle×10¹⁰/ml) Date (Temp.) AQF2-8 AQF2-9 AQF2-10 AQF2-11 AQF2-12 Sep. 25,1997 10.8 10.7 11.4 11.8 10.7 Nov. 05, 1997 (4° C.) 9.1 9.2 10.3 11.29.6 Nov. 05, 1997 (R.T.) 8.0 9.3 10.3 11.1 9.6 Dec. 12, 1997 (4° C.) 6.17.6 8.8 7.3 7.7 Dec. 17, 1997 (R.T.) 3.0 8.2 7.6 8.4 7.5 AqueousFormulation Set 2 Excipients AQF2-1 AQF2-2 AQF2-3 AQF2-4 AQF2-5 AQF2-6AQF2-7 mannitol (W %) 5 5 5 5 sucrose (W %) 5 5 5 5 glycine (M) 0.250.25 0.25 arginine (M) 0.25 0.25 urea (W %) 1 1 peg (w %) ExcipientsAQF2-8 AQF2-9 AQF2-10 AQF2-11 AQF2-12 mannitol (W %) 5 5 5 5 sucrose (W%) 5 5 5 5 10 glycine (M) 0.25 0.25 arginine (M) 0.25 0.25 urea (W %) 11 peg (w %) 1 1 Formulations are in 10 mM-tris buffer (pH = 7.5) whichconsists of 1% glycerol and 1 mM MgCl₂. The formulations are stored at4° C. and room temperature under nitrogen.

TABLE 14 Aqueous Formulation Set #3 HPLC Viral Particle PFU × 10⁹(×10⁹/ml) Date (temp.) F10-7 F10-8 F11-7 F11-8 F10-7 F10-8 F11-7 F11-8Oct. 3, 1997 2.2 3.3 2.1 2.8 12.1 12.0 11.8 12.0 Nov. 6, 3.4 4.0 2.8 3.410.6 10.5 10.1 10.3 1997 (−20° C.) Nov. 6, 3.5 3.6 4.3 2.8 10.0 9.7 9.910.3 1997 (4° C.) Jan. 15, 3.8 4.8 3.2 3.7 7.3 7.4 7.7 8.0 1998 (−20°C.) Jan. 15, 3.5 3.1 2.9 3.1 7.5 7.4 7.6 7.5 1998 (4° C.) ExcipientsF10-7 F10-8 F11-7 F11-8 mannitol (W %) 6 6 5 5 sucrose (VV %) 7 8 7 8HSA (W %) 0.5 0.5 0.5 0.5 glycerol (W %) 1 1 1 1 MgCl₂ (mM) 1 1 1 1

TABLE 15 Liquid formulation set #4 Date (Temp.) AQF4-1 AQF4-2 AQF4-3AQF4-4 AQF4-5 AQF4-6 AQF4-7 PFU (×10⁹/ml) Jan. 13, 1998 3.0 2.5 3.6 3.42.7 3.1 3.4 Feb. 16, 1998 (4° C.) 2.5 3.2 3.3 2.9 2.6 2.9 2.6 Feb. 16,1998 (R.T.) 1.8 2.7 1.6 3.6 2.6 1.6 1.7 Apr. 10, 1998 (4° C.) 2.2 2.02.6 3.0 2.4 1.9 2.2 4/10.98 (R.T.) 0.4 0.4 0.3 0.5 0.4 <0.1 1.1 Jul. 24,1998 (4° C.) 2.4 2.8 2.6 3.5 1.9 2.2 2.6 Jul. 24, 1998 (R.T.) 0.0020.005 0.006 0.005 0.005 0.005 0.001 Jan. 8, 1999 (4° C.) 2.9 2.4 2.1 2.62.0 2.2 2.1 Jan. 8, 1999 (R.T.) 0.0002 0.0004 0.0004 0.0002 0.00040.0004 0.00006 HPLC Viral Particles (×10¹⁰/ml) Jan. 13, 1998 7.2 8.8 9.29.0 7.8 7.9 9.1 Feb. 16, 1998 (4° C.) 7.5 9.3 9.2 9.5 8.2 8.4 9.6 Feb.16, 1998 (R.T.) 6.8 9.0 9.5 9.0 8.7 8.4 9.3 Apr. 10, 1998 (4° C.) 7.19.2 9.6 9.6 8.9 9.1 9.9 Apr. 10, 1998 (R.T.) 7.5 9.5 10.1 9.7 8.9 8.99.5 Jul. 24, 1998 (4° C.) 8.1 9.9 11.1 10.3 9.2 7.4 9.3 Jul. 24, 1998(R.T.) 7.3 3.0 10.7 8.9 10.4 10.45 3.5 Jan. 8, 1999 (4° C.) 7.8 10.310.3 10.1 8.7 1.7 9.5 Jan. 8, 1999 (R.T.) 8.4 11.0 11.3 11.0 9.7 10.49.4 Excipients AQF4-1 AQF4-2 AQF4-3 AQF4-4 AQF4-5 AQF4-6 AQF4-7 Mannitol(w %) 5 5 5 5 5 5 5 Sucrose (w %) 5 5 5 5 5 5 5 Tween-80 (w %) 0.02 0.10.5 Chap (w %) 0.02 0.1 0.5 Buffer: 10 mM Tris + H0.15M NaCl + 1mMMgCl2, pH = 8.2 Formulations were blanketed with N₂.

Table 10 and Table 11 show the storage stability data with secondarydrying at 30° C. without and with N₂ backfilling, respectively. Becauseof the nearly identical stability observed at −20° C. and 4° C. storageconditions, and to reduce the consumption of virus, −20° C. was notincluded in the long-term storage stability study. Similar to thesamples dried with secondary drying at 10° C., virus is stable at 4° C.but not stable at RT. However, relative better stability was observed atRT storage than those dried at 10° C. secondary drying. This is likelyto be the result of the lower residual moisture attained at 30° C.secondary drying. This result suggests that residual moisture is animportant parameter that affects storage stability during long termstorage.

HPLC viral particle recoveries are consistently lower than virusrecoveries calculated from PFU assay immediate after drying. The reasonfor the discrepancy is not clear. However, it is likely to be related topossible virus aggregation during freeze-drying. Electron microscopyevaluation is being carried out to examine possible virus aggregationafter lyophilization. During storage, HPLC analysis indicates that virusis stable at both −20° C. and 4° C. storage and not stable at RT, whichis consistent with the results from PFU assay.

Example 5 HSA Alternatives

The presence of HSA in the formulations could be a potential regulatoryconcern. As a result, a variety of excipients have been evaluated tosubstitute HSA in the formulation. The substitutes examined includedPEG, amino acids (glycine, arginine), polymers (polyvinylpyrrolidone),and surfactants (Tween-20 and Tween-80). These HSA substitutes are,however, suboptimal relative to HSA. Effort on further development wasminimal.

Example 6 Liquid Formulation

Concurrent with the development of lyophilization of Adp53 product,experimentation was carried out to examine the possibility of developinga liquid formulation for Adp53 product. The goal was to develop aformulation that can provide enough stability to the virus when storedat above freezing temperatures. Four sets of liquid formulations havebeen evaluated. In the first set of formulation, the current 10%glycerol formulation was compared to HSA and PEG containingformulations. In the second set of formulation, various amino acids wereexamined for formulating Adp53. In the third set of formulation, theoptimal formulation developed for lyophilization was used to formulateAdp53 in a liquid form. In the fourth set of formulation, detergentswere evaluated for formulating Adp53. Viruses formulated with all thosedifferent formulations are being tested for long term storage stabilityat −20° C., 4° C., and RT.

Liquid Formulation Set #1

HSA containing formulation (5% sucrose+5% HSA in 10 mM Tris buffer, 150mM NaCl, and 1 mM MgCl₂, pH=8.20 buffer) was compared with 10% glycerolin DPBS buffer and sucrose/PEG and Trehalose/PEG formulations. PEG hasbeen recommended as a good preferential exclusion agent in formulations(Wong and Parasrampurita, 1997). It is included in this set offormulation to examine whether it can provide stabilization effect onAdp53. Formulations were filled into the 3 ml lyo vials at a fill volumeof 0.5 ml. Vials were capped under either atmospheric or N₂ blanketingconditions to examine any positive effects N₂ blanketing may have onlong term storage stability of Adp53. To ensure adequate degassing fromthe formulation and subsequent N₂ blanketing, the filled vials waspartially stoppered with lyo stoppers and loaded onto the shelf of thelyophilizer under RT. The lyophilizer chamber was closed and vacuum wasestablished by turning on the vacuum pump. The chamber was evacuated to25 in Hg. Then the chamber was purged completely with dry N₂. Theevacuation and gassing were repeated twice to ensure complete N₂blanketing. N₂ blanketed vials were placed with the non-N₂ blanketedvials at various storage conditions for storage stability evaluation.Table 12 shows the analysis data for up to 18 months storage at 4° C.and RT.

Statistically significant drops in virus PFU and HPLC viral particleswere observed for 10% glycerol formulation after 3 months storage atboth 4° C. and RT. No statistically significant virus degradation wasobserved for all other formulations at 4° C. storage. However, decreasein virus infectivity was observed when stored at RT.

Liquid Formulation Set #2

Various combinations of amino acids, sugars, PEG and urea were evaluatedfor Adp53 stabilization during long storage. Table 13 shows the 12-monthstability data. The results indicate that combination of 5% mannitol and5% sucrose with other excipients gave better storage stability at RT forone month. Adp53 is most stable in formulation has all the excipients.In this set of formulation, no human or animal derived excipients wereincluded. It is our expectation to develop a liquid formulation withoutincluding any proteins derived from either human or animal origins.

Liquid Formulation Set #3

The optimal formulations developed for lyophilization was evaluated forformulating Adp53 in a liquid form. This approach would be a goodbridging between liquid formulation and lyophilization if satisfactoryAdp53 stability can be achieved using lyophilization formulation forliquid fill. Filled samples were stored at −20° C. and 4° C. forstability study. Table 14 shows the 3-month stability data. Virus isstable at both −20° C. and 4° C. for the four different formulations.This is in agreement with the results from formulation set #2, whichsuggests that better virus stability is expected with the presence ofboth mannitol and sucrose in the formulation. Longer time storagestability data is being accrued.

Liquid Formulation Set #4

Detergents have been used in the formulations for a variety ofrecombinant proteins. In this set of formulation, various concentrationsof detergents were examined for formulating Adp53. The detergents usedwere non-ionic (Tween-80) and zwitterionic (Chap). Table 15 shows the12-month stability data. Virus is stable at 4° C. storage. Nosignificant difference in virus stability at 4° C. was observed amongthe formulations tested. Similar to formulation set #2, no exogenousprotein is included in this set of formulation.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. An adenovirus liquid formulation comprising adenovirus, a polyol, anda buffer, wherein the polyol is present in an amount to promote themaintenance of adenovirus infectivity.
 2. The adenovirus liquidformulation of claim 1, wherein the adenovirus liquid formulationretains an infectivity of about 70% PFU/mL to about 99.9% PFU/mL of thestarting infectivity when stored for six months at 4 centigrade.
 3. Theadenovirus liquid formulation of claim 1, wherein the polyol is at aconcentration of from about 5% to about 30% (w/v).
 4. The adenovirusliquid formulation of claim 1, wherein the polyol is glycerol, propyleneglycol, polyethylene glycol, sorbitol, or mannitol.
 5. The adenovirusliquid formulation of claim 4, wherein the polyol is glycerol.
 6. Theadenovirus liquid formulation of claim 1, wherein the buffer is at aconcentration of from about 1 mM to 50 mM.
 7. The adenovirus liquidformulation of claim 6, wherein the buffer is at a concentration of fromabout 5 mM to about 20 mM.
 8. The adenovirus liquid formulation of claim1, wherein the buffer is Tris-HCl, TES, HEPES, mono-Tris, brucinetetrahydrate, EPPS, tricine, histidine, or PBS.
 9. The adenovirus liquidformulation of claim 1, wherein the buffer is Tris-HCl.
 10. Theadenovirus liquid formulation of claim 8, wherein the buffer is PBS. 11.The adenovirus liquid formulation of claim 1, wherein the polyol isglycerol and the buffer is Tris-HCl.
 12. The adenovirus liquidformulation of claim 1, wherein the polyol is glycerol and the buffer isPBS.
 13. The adenovirus liquid formulation of claim 1, wherein thepolyol is at a concentration of from about 5% to about 30% (w/v) and thebuffer is at a concentration of from about 5 mM to about 20 mM.
 14. Theadenovirus liquid formulation of claim 13, wherein the polyol isglycerol and the buffer is Tris-HCl.
 15. The adenovirus liquidformulation of claim 13, wherein the polyol is glycerol and the bufferis PBS.
 16. An adenovirus liquid formulation comprising adenovirus, apolyol, a buffer, and a non-ionic detergent, wherein the polyol ispresent in an amount to promote the maintenance of adenovirusinfectivity.
 17. The adenovirus liquid formulation of claim 16, whereinthe non-ionic detergent is Tween-20 or Tween-80.
 18. The adenovirusliquid formulation of claim 17, wherein the non-ionic detergent isTween-80.
 19. The adenovirus liquid formulation of claim 18, wherein thepolyol is glycerol.
 20. The adenovirus liquid formulation of claim 19,wherein the buffer is Tris-HCl.
 21. The adenovirus liquid formulation ofclaim 19, wherein the buffer is PBS.
 22. An adenovirus liquidformulation comprising adenovirus, a polyol, a buffer, and a saltselected from the group consisting of MgCl₂, MnCl₂, CaCl₂, ZnCl₂, NaCl,and KCl, wherein the polyol is present in an amount to promote themaintenance of adenovirus infectivity.
 23. The adenovirus liquidformulation of claim 22, wherein the salt is MgCl₂.